Conjugates of biologically active compounds, methods for their preparation and use, formulation and pharmaceutical applications thereof

ABSTRACT

This invention features a method of identifying a compound useful for enhancing efficacy of a therapeutic agent. The method includes incubating a compound in blood cells; separating immune cells from erythrocytic cells; and determining the ratio of the concentration of the compound in the immune cells to the concentration of the compound in the erythrocytic cells; wherein the compound comprises a transportophore and a therapeutic agent, in which the transportophore is covalently bonded to the therapeutic agent via a bond or a linker.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/367,624, filed Feb. 14, 2003, pending, which claims benefit of U.S.patent application Ser. No. 60/357,589, filed Feb. 15, 2003. Thecontents of each of these patent applications is hereby incorporated byreference.

BACKGROUND

Successful therapy with a pharmaceutical agent requires that the agentsatisfy numerous requirements imposed by the physiology of the host andof the disease or condition. The requirements include: (i) adequateability to interact with the target receptor(s); (ii) appropriatephysical properties for presence at the location of the receptors inconcentrations that permit the interactions noted above; (iii)appropriate physical properties to allow the agent to enter the body anddistribute to the location of the receptors by any means; (iv)sufficient stability in fluids of the body; (v) the absence of toxiceffects in compartments where the therapeutic agent is mostconcentrated, or in any other compartment where the therapeutic agent islocated; and (vi) the absence of sequestration into non-physiologicalcompartments and so on.

In general, these compounding requirements limit the nature ofpharmaceutical compounds that have utility in vivo and thus reduce theprobability of discovering adequately active molecules from de novostarting points. In response to these constraints, significant efforthas been applied to the question of predicting ideal physical propertiesfor pharmaceutical molecules. Authors such as Lipinski (Lipinski et al.,2001) have described rules of therapeutic agent design which, amongstother parameters, predicts that ideal therapeutic agents will have fewfunctions such as hydroxy groups, a molecular weight below 500 Da, mildbasicity, and moderate lipophilicity (logP<5) (Lipinski et al., 2001).Unfortunately, these parameters are too general to inform the directsynthesis of highly bioavailable compounds. Furthermore, theserequirements are not helpful for larger molecule chemistry (MW>500) suchas the compounds disclosed here.

Recently, improvements in the technology of synthetic chemistry andmolecular biology have allowed the testing of large numbers of moleculesand the discovery of many ligands with adequate affinity to theirtargets to have some potential in vivo. Many such molecules proveinadequate on in vivo testing largely due to the manifold, stringent,and often conflicting (i.e. stability without toxicity) requirementsoutlined above.

In addition to the difficulties facing many new molecules, many existingmolecules in clinical use also exhibit inadequate properties of uptake,distribution, stability and toxicity (Lipinski et al. 2001). Theseobservations demonstrate, that in general, deficiencies in uptake,distribution, and stability result in inadequate therapy from existingmolecules and inadequate and uneconomical probabilities of success inthe discovery of new molecules.

Such problems often fall within the scope of therapeutic agentdelivery—a discipline which combines many aspects of formulation withtechniques for introducing the agent into the host body. Deliverymethods are frequently designed to permit passage through a singlebarrier (i.e. the skin) (WO 01/13957) or the intestine (WO 01/20331)after which the agent must again conform with the general requirementsabove in order to act at the in vivo target. Certain delivery strategiesinvolve a physical preparation such as liposomes (Debs et al. 1990;Jaafari, Foldvari, 2002) or anti-body conjugates (Everts et al., 2002)which further direct the molecules within the host body. Others rely onthe addition of cationic lipids to formulations, the use of transportproteins as a route of uptake (WO 01/20331). The use of transportprocesses deliberately in therapeutic agent design is perhaps bestillustrated by the nucleoside therapeutic agents, which to varyingdegrees, are taken up as metabolites and whose transport to mitochondriais a major cause of toxicity (WO 98/29437) For example, see EuropeanPatent No. 0009944B1, European Patent No. 0044090A3, and Japanese PatentNo. 05163293. Such methods may enhance performance in therapy or reducetoxicity but they increase cost and require direct introduction into theblood stream which is impractical in chronic use.

More preferable would be small molecules that possess the appropriatestructures and properties to mediate efficient uptake and stability.Such small molecules would ideally be able to carry a range oftherapeutic agents of varying properties such that they could becommercialized in more than one indication. However, there is arequirement that they be inactive and stable enough to ensure that thecargo molecule is carried in the periphery (Harada et al. 2000).

The present invention represents a significant advance in that itprovides for a means of improving the bioavailability and efficacy of avariety of molecules in vivo using a series of rational and facileassays to select desirable compounds based on known pharmacophores orpharmaceutical lead structures that have not been optimized for in vivoaction.

SUMMARY

The invention relates to a compound useful for enhancing efficacy of atherapeutic agent, a method for identifying such a compound, and amethod of treating diseases including inflammation, graft rejection,infection, cancer, allergies, metabolic cardiovascular, pulmonary,dermatological, rheumatological and hepatic diseases. The inventionfurther comprises compositions and formulations selected using themethod and applications for same.

The invention provides for a method for identifying compounds that actas carriers or “transportophores” (i.e., a transport mediating molecule)that when combined, either directly or via a linker, to a wide varietyof therapeutic agents, improves one or more of the followingcharacteristics of the agent: ease of formulation, gastric stability,bioavailability, stability, disposition, elimination, half life,efficacy, safety, duration of action and selectivity.

In one aspect, this invention features a compound of the followingformula (or referred to as T-L-C hereinafter):

TL-C)_(m),

wherein T is a transportophore, L is a bond or a linker having amolecular weight up to 240 dalton, C is a non-antibiotic therapeuticagent, and m is 1, 2, 3, 4, 5, 6, 7, or 8, in which the transportophorehas an immune selectivity ratio of at least 2, the transportophore iscovalently bonded to the non-antibiotic therapeutic agent via the bondor the linker, and the compound has an immune selectivity ratio of atleast 2. Note that when there are more than one L or C moieties (i.e., mis greater than 1), the L moieties or the C moieties, independently, canbe the same or different. The same rule applies to other similarsituations.

The transportophore can be a metabolite, a natural product, a metabolitemimic, a metabolite derivative (e.g., a sugar, amino, or peptidederivative), a fatty acid, a bile acid, a vitamin, a nucleobase, analcohol, or an organic acid or base, a portion of which resembles and isrecognized as a substrate for transport protein(s). It can be anamphiphilic molecule having a pKa value of 6.5 to 9.5, or a cyclic orheterocyclic molecule (e.g., lactone, lactam, ether, cyclic acetal orhemi-acetal). The cyclic or heterocyclic molecule can have an attachedsugar. The cyclic or heterocyclic molecule can be a macrolactone ormacroether, including a macrolactone or macroether having an attachedsugar. The cyclic or heterocyclic molecule can also be a macrolide orketolide having an amino sugar, including a macrolide having mono-, di-,or tri-basic groups (e.g., an amine). In some embodiments, the macrolidehas no intrinsic antibacterial activity (inactive at 50 uM or higherconcentrations when tested against Bacillus in vitro see protocol) and apKa value of less than 9.0 (e.g., 8.5, 8.0, 7.5, 7.0, or any number inbetween).

In some embodiments, the compound has the following formula (In which abond, drawn without any attached groups, means a methyl group. The samerule applies to other similar situations):

Wherein,

X=N(R⁷)—CH₂

-   -   CH₂—N(R⁷)    -   C(═O)    -   C(═NOR⁸)    -   CH(OR⁹)    -   CH(NR¹⁰R¹¹)    -   C(═NR¹²)    -   OC(═O)    -   C(═O)O

Y=independently,

-   -   Linker (as defined below)

Z=C(═O)—

-   -   CH(R¹⁶)

R¹=H

-   -   CH₃    -   (C₂-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹    -   Y—R¹³    -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵

R²=H

-   -   (1′,2′-cis)-OH    -   (1′,2′-trans)-OH    -   (1′,2′-cis)-OR¹⁵    -   (1′,2′-trans)-OR¹⁵    -   (1′,2′-cis)-SH    -   (1′,2′-cis)-S—Y—R¹³    -   or the R¹ and R² bearing atoms are connected via a —OC(═O)CHR¹⁶—        element

R³=H

-   -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵

R⁴=H

-   -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵

R⁵=H

-   -   or R⁴, R⁵ are connected by Z

R⁶=H

-   -   CH₃

R⁷=H

-   -   CH₃    -   Y—R¹³    -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵

R⁸=H

-   -   Y—R¹³    -   R¹³    -   C(═O)—R¹⁷    -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹

wherein alkyl, alkenyl, alkynyl, aryl, and heteroaryl groups areoptionally substituted by one to five substituents selectedindependently from halogen, (C₁-C₄)alkyl, (C₁-C₄)alkenyl,(C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl,(C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, hydroxy, nitro, cyano,azido, mercapto, —NR¹⁸R¹⁹, R¹⁸C(═O)—, R¹⁸C(═O)O—, R¹⁸C(═O)O—,R¹⁸NHC(═O)—, R¹⁸C(═O)NH—, R¹⁸R¹⁹NC(═O)— and R¹⁸OC(═O)—

R⁹=H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl        wherein alkyl, alkenyl, alkynyl, aryl, and heteroaryl groups are        optionally substituted by one to five substituents selected        independently from halogen, (C₁-C₄)alkyl, (C₁-C₄)alkenyl,        (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl,        (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, hydroxy, nitro,        cyano, azido, mercapto, —NR¹⁸R¹⁹, R¹⁸C(═O)—, R¹⁸C(═O)O—,        R¹⁸OC(═O)O—, R¹⁸NHC(═O)—, R¹⁸C(═O)NH—, R¹⁸R¹⁹NC(═O)— and        R¹⁸OC(═O)—

R¹⁰, R¹¹=independently H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)akynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹    -   or R¹⁰=H and R¹¹=—Y—R¹³    -   C(═O)—Y—R¹⁵, —C(═O)—R¹⁵

R¹²=H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹    -   Y—R¹³

R¹³=R¹⁵=independently, therapeutic agent

R¹⁶=H

-   -   CH₃    -   (C₂-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹    -   Y—R¹³,

R¹⁷=O—R²⁰-aryl

-   -   optionally substituted by —X′—Y— therapeutic agent, X′—        therapeutic agent wherein X′ is    -   O    -   NH

R¹⁸, R¹⁹=independently H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl

R²⁰=independently

-   -   Halogen    -   (C₁-C₃)alkyl    -   NO₂    -   CN    -   OCH₃    -   N(CH₃)₂    -   N₃    -   SH    -   S(C₁-C₄)alkyl

In some other embodiments, the compound has the following formula:

Wherein,

X=N(R⁷)—CH₂

-   -   CH₂—N(R⁷)    -   C(═O)    -   C(═NOR⁸)    -   CH(OR⁹)    -   CH(NR¹⁰R¹¹)    -   C(═NR¹²)    -   OC(═O)    -   C(═O)O

Y=independently, Linker (as defined below)

Z=C(═O)—

-   -   CH(R¹⁶)—

R¹=H

-   -   CH₃    -   (C₂-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹    -   Y—R¹³    -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵    -   S(═O)_(k)(C₁-C₁₀)alkyl    -   S(═O)_(k)(C₁-C₁₀)alkenyl    -   S(═O)_(k)(C₁-C₁₀)alkynyl    -   S(═O)_(k)(C₆-C₁₀)aryl    -   S(═O)_(k)(C₂-C₉)heteroaryl    -   S(═O)_(k)—Y—R¹⁵    -   S(═O)_(k)—R¹⁵        wherein k is 0, 1 or 2, and alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl and heteroaryl can optionally be        substituted by one to three halogen, cyano, hydroxy,        (C₁-C₄)alkyloxy, nitro, (C₁-C₆)alkyl, (C₁-C₆)alkenyl,        (C₁-C₆)alkynyl, (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl,        (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, NR¹⁸R¹⁹, R¹⁸C(═O)—, R¹⁸C(═O)O—,        R¹⁸OC(═O)—, R¹⁸C(═O)NH—, R¹⁸NHC(═O)—, R¹⁸R¹⁹NC(═O)— and        R¹⁸OC(═O)—O—    -   R²=H    -   (1′,2′-cis)-OH    -   (1′,2′-trans)-OH    -   (1′,2′-cis)-OR¹⁵    -   (1′,2′-trans)-OR¹⁵    -   (1′,2′-cis)-SH    -   (1′,2′-cis)-S—Y—R¹³    -   or the R¹ and R² bearing atoms are connected via a —OC(═O)CHR¹⁶—        element

R^(3a), R^(3b)=independently H

-   -   R¹    -   OH    -   OR¹¹    -   NR¹⁰R¹¹

or R^(3a)=R^(3b)=(═O),

-   -   (═NR¹)    -   O(CH₂)_(k)O— wherein k is 2 or 3

R⁴=H

-   -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵

R⁵=H

or R⁴, R⁵ are connected by -Z-

R⁶=H

-   -   CH₃

R⁷=H

-   -   CH₃    -   Y—R¹³    -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵

R⁸=H

-   -   Y—R¹³    -   C(═O)—R¹⁷

R⁹=H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl

R¹⁰, R¹¹=independently H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)akynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₂-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl are optionally substituted by one to three        halogen, cyano, hydroxy, (C₁-C₄)alkyloxy, nitro, (C₁-C₆)alkyl,        (C₁-C₆)alkenyl, (C₁-C₆)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        NR¹⁸R¹⁹, R¹⁸C(═O)—, R¹⁸C(═O)O—, R¹⁸OC(═O)—, R¹⁸C(═O)NH—,        R¹⁸NHC(═O)—, R¹⁸R¹⁹NC(═O)— and R¹⁸OC(═O)—O—

or R¹⁰=H and

R¹¹=Y—R¹³

-   -   C(═O)—Y—R¹⁵    -   C(═O)—R¹⁵    -   S(═O)_(k)(C₁-C₁₀)alkyl    -   S(═O)_(k)(C₁-C₁₀)alkenyl    -   S(═O)_(k)(C₁-C₁₀)alkynyl    -   S(═O)_(k)(C₆-C₁₀)aryl    -   S(═O)_(k)(C₂-C₉)heteroaryl    -   S(═O)_(k)—Y—R¹⁵    -   S(═O)_(k)—R¹⁵        wherein k is 0, 1 or 2 and alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl and heteroaryl can be substituted as        defined above.

R¹²=H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹    -   Y—R¹³

R¹³=R¹⁵=independently therapeutic agent

R¹⁶=H

-   -   CH₃    -   (C₂-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl.    -   (C₁-C₄)alkyliden-NR¹⁸R¹⁹    -   Y—R¹³

R¹⁷=O—R²⁰-aryl

-   -   optionally substituted by —X′—Y— therapeutic agent, X′—        therapeutic agent wherein X′ is    -   S, O, NH

R¹⁸, R¹⁹=independently H

-   -   (C₁-C₁₀)alkyl    -   (C₁-C₁₀)alkenyl    -   (C₁-C₁₀)alkynyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkyl    -   (C₁-C₈)[(C₁-C₄)alkoxy]alkenyl    -   (C₆-C₁₀)aryl-(C₁-C₅)alkyl    -   (C₂-C₉)heteroaryl-(C₁-C₅)alkyl

R²⁰=independently,

-   -   Halogen    -   (C₁-C₃)alkyl    -   NO₂    -   CN    -   OCH₃    -   N(CH₃)₂    -   N₃    -   SH    -   S(C₁-C₄)alkyl

In still some other embodiments, the compound has the following formula:

Wherein,

X=N(R⁹)—CH₂

-   -   CH₂—N(R⁹)    -   C(═O)    -   C(═NOR¹⁰)    -   C(OR¹¹)H    -   CH(NR¹²R¹³)    -   C(═NR¹⁴)    -   OC(═O)    -   C(═O)O

Y=independently, Linker (as defined below)

R¹=OR¹⁷

-   -   NR¹⁷R¹⁸    -   or R¹ is connected to the oxygen bearing R⁴ or R⁵ forming a        lactone or is connected to a suitable substituent in R² forming        a lactone or lactam.

R²=O-2-cladinosyl

-   -   H    -   X′, wherein X′=halogen    -   azido    -   nitro    -   cyano    -   OR¹⁷    -   OR²²    -   NR¹⁷R¹⁸    -   SR¹⁷(C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—, —Y-therapeutic agent or        -therapeutic agent

R³=H

-   -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, R²⁰R²¹N—

R⁴=O-2-desosaminyl

-   -   H    -   C(═O)R¹⁷    -   Y-therapeutic agent    -   therapeutic agent    -   S(═O)₂R¹⁷ providing R¹⁷ is not hydrogen    -   C(═O)NR¹⁷R¹⁸ (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—, —Y-therapeutic agent or        -therapeutic agent        or R⁴ is connected to a suitable R² containing a N or a O by        —C(═O), S(═O)_(n) wherein n=1 or 2, —CR²⁰R¹⁷—,        CR²⁰(—Y-therapeutic agent)-, —CR²⁰(-therapeutic agent)- forming        in dependence of R² a 6 or 7-membered ring

R⁵=R²⁰

-   -   C(═O)R²⁰    -   or R⁴, R⁵ are connected by C(═O), S(═O)_(n) wherein n=1 or 2,        —CR²⁰R¹⁷—, CR²⁰(—Y—therapeutic agent)-, —CR²⁰(-therapeutic        agent)-

R⁶, R⁸=independently H

-   -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—, —Y-therapeutic agent or        -therapeutic agent,

or R⁶, R⁸=independently —C(═O)R¹⁷, —Y-therapeutic agent, -therapeuticagent, —S(═O)₂R¹⁷ providing R¹⁷ is not hydrogen, —C(═O)NR¹⁷R¹⁸

R⁷=H

-   -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—, —Y-therapeutic agent or        -therapeutic agent        or two of each R⁶, R⁷, R⁸ are connected by —C(═O), S(═O)_(n)        wherein n=1 or 2, —CR²⁰R¹⁷—, CR²⁰(—Y-therapeutic agent)-,        —CR²⁰(-therapeutic agent)-

R⁹=H

-   -   CH₃    -   Y-therapeutic agent    -   therapeutic agent    -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl,        wherein alkyl, alkenyl, alkynyl groups are optionally        substituted by one to five substituents selected independently        from halogen, (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl,        (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl,        (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido,        mercapto, R²⁰R²¹N—, R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—,        R²⁰NHC(═O)—, R²⁰C(═O)NH—, R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—,        —Y-therapeutic agent or -therapeutic agent

R¹⁰=C(═O)-aryl

-   -   therapeutic agent    -   H    -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl,        wherein alkyl, alkenyl, alkynyl groups are optionally        substituted by one to five substituents selected independently        from halogen, (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl,        (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl,        (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido,        mercapto, R²⁰R²¹N—, R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—,        R²⁰NHC(═O)—, R²⁰C(═O)NH—, R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—,        —Y-therapeutic agent or -therapeutic agent

R¹¹=H

-   -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl,        wherein alkyl, alkenyl, alkynyl groups are optionally        substituted by one to five substituents selected independently        from halogen, (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl,        (C₃-C₇)cycloalkyl, (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl,        (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido,        mercapto, R²⁰R²¹N—, R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—,        R²⁰NHC(═O)—, R²⁰C(═O)NH—, R²⁰R²¹NC(═O)—, R²⁰OC(═O)O—,        —Y-therapeutic agent or -therapeutic agent,

or R¹¹=—Y-therapeutic agent, -therapeutic agent, —C(═O)R¹⁷

R¹², R¹³=independently H

-   -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl,        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, R²⁰OC(═O)O—, —Y-therapeutic agent or -therapeutic        agent,    -   or R¹², R¹³=independently —C(═O)R¹⁷, —Y-therapeutic agent,        -therapeutic agent, —S(═O)₂R¹⁷ providing R¹⁷ is not hydrogen,        —C(═O)NR¹⁷R¹⁸

R⁴=independently

-   -   therapeutic agent    -   H    -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, R²⁰OC(═O)O—, —Y-therapeutic agent or -therapeutic        agent

R¹⁵=independently

-   -   H    -   C(═O)R¹⁷    -   Y-therapeutic agent    -   therapeutic agent    -   S(═O)₂R¹⁷ providing R¹⁷ is not hydrogen    -   C(═O)NR¹⁷R¹⁸    -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl,        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—, —Y-therapeutic agent or        -therapeutic agent

R¹⁶=H

-   -   OR¹⁷    -   OR²²

R¹⁷, R¹⁸=independently H

-   -   (C₁-C₆)alkyl    -   (C₁-C₆)alkenyl    -   (C₁-C₆)alkynyl    -   (C₃-C₁₀)cycloalkyl    -   (C₁-C₉)heterocycloalkyl    -   (C₆-C₁₀)aryl    -   (C₁-C₉)heteroaryl        wherein alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,        aryl, heteroaryl groups are optionally substituted by one to        five substituents selected independently from halogen,        (C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,        (C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl,        (C₁-C₄)alkoxy, hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—,        R²⁰C(═O)—, R²⁰C(═O)O—, R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—,        R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—, —Y-therapeutic agent or        -therapeutic agent

or provided that connected to a nitrogen, R¹⁷, R¹⁸ may form a cyclicstructure of 4 to 7 members (including the nitrogen). R¹⁷ and R¹⁸ thencan represent a fragment from the type of-[C(AB)]_(m)-Ξ_(n)-[C(DE)]_(o)-Ψ_(p)-[C(GJ)]_(q) wherein m, n, o, p andq independently are 0, 1, 2, 3, 4, 5, or 6, Ξ and Ψ independently are—O—, —S—, —NK— and A, B, D, E, G, J, and K independently are hydrogen,(C₁-C₄) alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₃-C₇)cycloalkyl,(C₁-C₆)heterocycloalkyl, (C₆-C₁₀)aryl, (C₁-C₉)heteroaryl, (C₁-C₄)alkoxy,hydroxy, nitro, cyano, azido, mercapto, R²⁰R²¹N—, R²⁰C(═O)—, R²⁰C(═O)O—,R²⁰OC(═O)—, R²⁰NHC(═O)—, R²⁰C(═O)NH—, R²⁰R²¹NC(═O)—, and R²⁰OC(═O)O—

R²⁰, R²¹=independently H

-   -   (C₁-C₆)alkyl

R²²=independently

-   -   C(═O)R¹⁷    -   Y-therapeutic agent    -   therapeutic agent    -   S(═O)₂R¹⁷ providing R¹⁷ is not hydrogen, —C(═O)NR¹⁷R¹⁸.

In further embodiments, the compound has the following formula:

Wherein

m=independently, 0, 1, 2, 3

n=0-7

X=independently

-   -   O    -   S    -   Se    -   NR¹    -   PR¹

with the proviso, that at least one X=—NR¹—

A=independently

-   -   CH₂    -   CHR²    -   CR²R³    -   C(═O)

with the proviso, that at least one X=—NR¹— is not an amide

R¹=independently,

-   -   H    -   (C₁-C₁₀)alkyl optionally substituted by fluoro, cyano, R⁴,        R⁴O₂C, R⁴C(═O)NH and R⁴S(═O)_(k) wherein k is 0, 1 or 2    -   R⁴C(═O), R⁴S(═O)_(k) wherein k is 0, 1 or 2

R², R³=independently NH₂

-   -   NHR¹    -   NR¹R⁵    -   OH,    -   OR⁴    -   R⁴C(═O) (C₁-C₆)alkyl    -   (C₂-C₁₂)alkenyl    -   (C₂-C₁₂)alkynyl    -   (C₃-C₁₀)cycloalkyl(C₁-C₆)alkyl    -   (C₂-C₉)heterocycloalkyl(C₁-C₆)alkyl    -   (C₆-C₁₀)aryl(C₁-C₆)alkyl    -   (C₂-C₉)heteroaryl(C₁-C₆)alkyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, and heteroaryl groups are optionally        substituted by one to three halo, (C₁-C₄)alkoxy, hydroxy, nitro,        cyano, —C(═O)—OR⁸, —C(═O)N(H)R⁸, (C₆-C₁₀)aryl,        (C₂-C₉)heteroaryl, N*R⁵R⁶R⁷ wherein * is no or a positive        charge, one or two of R², R³ can be a directly coupled        therapeutic agent

R⁴=independently

-   -   NH₂    -   NHR⁹    -   NR⁹R⁵    -   OH    -   OR⁹    -   (C₁-C₆)alkyl    -   (C₂-C₁₂)alkenyl    -   (C₂-C₁₂)alkynyl    -   (C₃-C₁₀)cycloalkyl(C₁-C₆)alkyl    -   (C₂-C₉)heterocycloalkyl(C₁-C₆)alkyl    -   (C₆-C₁₀)aryl(C₁-C₆)alkyl    -   (C₂-C₉)heteroaryl(C₁-C₆)alkyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, and heteroaryl groups are optionally        substituted by one to three halo, (C₁-C₄)alkoxy, hydroxy, nitro,        cyano, R⁸, —C(═O)—OR⁸, —C(═O)N(H)R⁸, (C₆-C₁₀)aryl,        (C₂-C₉)heteroaryl, N*R⁵R⁶R⁷ wherein * is no or a positive        charge, or therapeutic agent

R⁵, R⁶=independently H

-   -   (C₁-C₆), optionally substituted by hydroxy    -   (C₆-C₁₀)aryl    -   (C₂-C₉)heteroaryl

R⁷=independently

-   -   lone electron pair    -   CH₃    -   C₂H₅    -   C₃H₇    -   CH₂—C₆H₅

R⁸=independently,

-   -   therapeutic agent

R⁹=independently,

-   -   (C₁-C₆) alkyl    -   (C₂-C₁₂)alkenyl    -   (C₂-C₁₂)alkynyl    -   (C₃-C₁₀)cycloalkyl(C₁-C₆)alkyl    -   (C₂-C₉)heterocycloalkyl(C₁-C₆)alkyl    -   (C₆-C₁₀)aryl(C₁-C₆)alkyl or    -   (C₂-C₉)heteroaryl(C₁-C₆)alkyl,        wherein the alkyl, alkenyl, alkynyl, cycloalkyl,        heterocycloalkyl, aryl, and heteroaryl groups are optionally        substituted by one to three halo, (C₁-C₄)alkoxy, hydroxy, nitro,        cyano, R⁸, —C(═O)—OR⁸, —C(═O)N(H)R⁸, (C₆-C₁₀)aryl,        (C₂-C₉)heteroaryl, N*R⁵R⁶R⁷ wherein * is no or a positive        charge, or therapeutic agent.

Preferred molecules can be compounds that are recognized by a transportenzyme in the membrane of the cell of the tissue that is to target. Thiscan be a molecule that fulfills the structural requirements in order tobe recognized by an oligo-peptide transporter.

Compounds recognized by transport enzymes can be identified byperforming a transport assay with the compound in question in cellsexpressing the transport protein in question, and comparing the level ofcompound accumulation with those from parallel uptake assays performedusing cells which do not express the target transport protein.

According to well known models these structures may be as exemplified inthe following sketches:

In these examples R (including R₁ and R₂) may represent a chemicalresidue that will modify the recognition by the transporting enzyme orat least not inhibit it. R may be comprised of the therapeutic agentthat is to be delivered or the pharmaceutical entity is for example anamino acid itself as in example A.

Necessary for transport through an oligopeptide transporter seems to bea basic group spaced 4 or 5 bonds from an hydrogen bond accepting grouplike preferably carboxylate (example A-C) or less preferred amide(example D).

EXAMPLE A

R₁ and R₂ are hydrogen or lower alkyl, branched or linear from C₁ to C₅,or benzyl or p-hydroxy benzyl, or hydroxy or mercapto methyl, or anygroup responsible for the desired pharmacological effect.

EXAMPLE B

R can be the moiety responsible for the pharmacological effect, or thepharmacologically relevant group linked on the carbon chain by achemical linker like an amide (amido-R═NH(C═O)—R′ (R′=pharmacologicallyrelevant group)).

EXAMPLE C

R can be the moiety responsible for the pharmacological effect, or thepharmacologically relevant group linked on the carbon chain by achemical linker like an amide (amido R═NH(C═O)—R′ (R′=pharmacologicallyrelevant group)).

EXAMPLE D

R2 can be hydrogen or lower alkyl, branched or linear from C1 to C5, orbenzyl or p-hydroxy benzyl, or hydroxy or mercapto methyl, while R1consists of the pharmacologically relevant therapeutic agent. Preferablythe therapeutic agent would contain a carboxylic acid that by linking tothe amino function of an amino acid hydrazide would obtain the generalstructure of example D.

Therapeutic agents and Transportophores can be directly connected or viaa linking element. This element typically is a bifunctional molecule oflow molecular mass, which can react subsequently with the therapeuticagent and the transportophore. Ideally the therapeutic agent can bereleased from this linker under physiological conditions. This may beachieved oxidatively (i.e. by action of a cytochrome C), reductively(i.e. by action of NADH), hydrolytically (i.e. by action of a protease),or initiated by radicals (i.e. by the action of superoxide radicals).The mechanisms of therapeutic agent release are not limited to the aboveexamples.

Linkers have the following formula:

F¹-M-F²

Where can be:

F¹, F²=independently a functional group, suitable to react with acounterpart in the therapeutic agent or in the transportophore. F¹ andF² are, but are not limited to

X¹ wherein X¹ is a halogen atom or a sulfonate ester or another suitableleaving group;

C(═O)X² wherein X² is Cl, Br or I,

CHO;

C(═O)OR^(a) wherein R^(a) is (C₁-C₄)alkyl or aryl, optionallysubstituted by 1-5 halogen atoms;

C(═O)OC(═O)OR^(b) wherein R^(b) is (C₁-C₅)alkyl or (C₁-C₅)alkenyl;

OH;

NHR^(c) wherein R^(c) is H, (C₁-C₄)alkyl;

NCX³ wherein X³ is S or O;

C(═O)CR═CHR′, wherein R and R′ are independently —H, —CH₃, —Cl, —Br, —F,—O(C₁-C₄)alkyl, —C(═O)O(C₁-C₄)alkyl, —NO₂, —S(═O)_(k)(O)_(l)(C₁-C₄)alkylwherein k is 0, 1 or 2 and 1 is 0 or 1, SiR¹R²R³ wherein R¹, R² and R³independently are (C₁-C₄)alkyl;

SX⁴ wherein X⁴ is —H, —Cl, —S_(k)(C₁-C₄)alkyl, S_(k)(C₆-C₁₀)aryl whereink is 1 or 2.

F¹ and F² can be connected to form a cyclic anhydride or di- ortrisulfide.

M is a spacing element which is, but is not limited to

(C₁-C₈)alkyl,

(C₁-C₈)alkenyl,

(C₁-C₈)alkynyl,

(C₃-C₁₀)cycloalkyl,

(C₆-C₁₀)aryl,

(C₂-C₉)heteroalkyl,

(C₂-C₉)heteroaryl.

Alkyl-, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl spacingelements are optionally substituted by (C₁-C₆)alkyl, 1-4 halogens,(C₁-C₄)alkoxy, (C₁-C₄)alkoxycarbonyl, hydroxy, amino, (C₁-C₄)alkylamino,(C₁-C₄)dialkylamino, (C₃-C₁₀)cycloalkyl, (C₁-C₆)alkylcarbonyloxy,(C₁-C₆)alkylcarbonylamido, (C₁-C₄)alkylamidocarbonyl,(C₁-C₄)dialkylamidocarbonyl, nitro, cyano, (C₁-C₄)alkylimino, mercaptoand (C₁-C₄)alkylmercapto functions.

TABLE 1 Non-limiting examples of Linkers useful in the synthesis ofT-L-C molecules.* Donor linking Recipient linking function function COOHNH2 OH COOH 1. Ethylendiamine, N-Methoxycarbonyl-4- 2. N- Glycol,hydroxyproline, Methoxycarbonyl-4- (2-Aminoethyl)-(2- Glycolic acid,β-Alanin, hydroxyproline, hydroxyethyl)amino β-hydroxy propanoicGlycolic acid, acid β-Alanin, β-hydroxy propanoic acid NH2 3. N-Ethylendiamine, 4. 2,2- Methoxycarbonyl-4- 2,2-DimethylsuccinicDimethylsuccinic acid, hydroxyproline, acid, Succinic acid, GlutaricGlycolic acid, Succinic acid, Glutaric acid, β-Alanin, β-hydroxy acid,2,4- 2,4-Dimethylglutaric propanoic acid Dimethylglutaric acid, acid,Methyl Methyl dicarboxy- dicarboxymethylamino methylamin,2-Aminoethyl-2- hydroxyethylamino OH 5. N- 2,2-Dimethylsuccinic 6.β-Hydroxy Methoxycarbonyl-4- acid, Succinic acid, propanoic acid,hydroxyproline, Glutaric acid, 2,2-Dimethylsuccinic Glycolic acid,2,4-Dimethylglutaric acid, Succinic acid, β-Alanin, acid, Glutaric acid,β-hydroxy propanoic Methyl dicarboxy- 2,4-Dimethylglutaric acidmethylamin, acid, 2-Aminoethyl-2- Methyl hydroxyethylaminodicarboxymethylamino 7. *The donor linking function in vertical refersto a functional group on T; the recipient linking function in horizonalrefers to a functional group on L; and the chemical groups in the boxesare the linkers (L).

The non-antibiotic therapeutic agent can be an anti-inflammatory agent,an anti-infectious agent (including anti-virals), an anti-cancer agent,an allergy-suppressive agent, an immune-suppressant agent, an agent fortreating a hematopoietic disorder, a lipid lowering agent, an agent fortreating a lysosomal storage disorder, a sterol synthesis modifyingagent, agents active on protozoa, or an agent for treating a metabolicdisease.

As used herein, an “immune selectivity ratio” is the ratio of theconcentration of a compound in immune cells (e.g., neutrophils,monocytes, and lymphocytes) to the concentration of the compound inerythrocytic cells after the compound has been incubated in a mixture ofblood cells including erythrocytes. A protocol of determining the immuneselectivity ratio is described in Example 1.

A “therapeutic agent,” as used herein, is a molecule withpharmacological activity (e.g., a therapeutic agent, medicine,medicament, or active agent), a disease modification agent, or any othermolecule that can be covalently attached to a transportophore via a bondor a linker which may have a desirable mode of action in immune ortarget cells. A therapeutic agent may be released from a compounddescribed above in response to the enzyme activity or thephysicochemical environment of the targeted cells. Thus, the therapeuticagent is selectively accumulated in a cell due to specificcharacteristics of the cell membranes, specific expression of membraneproteins, specific conditions within the cell, notably to expression ofspecific proteins such as granule proteins, binding sites in thecytoplasm, or other membrane bound or soluble proteins, and is thustrapped in the cell and therefore exhibits an enhanced or desiredactivity therein.

An “amphiphilic molecule,” as used herein, is a molecule having ahydrophilic (polar) and hydrophobic (non-polar) functional groups (e.g.,atoms) or a combination of groups (or atoms). The pKa of this moleculeis in the range of 6.5 to 9.5.

The term “cyclic” refers to a hydrocarbon cyclic ring including fullysaturated, partially saturated, and unsaturated mono-, bi-, andtri-cyclic rings having 4 to 34 ring atoms, preferably, 7 to 10, or 10to 15 ring atoms. The term “heterocyclic” refers to a hydrocarbon cyclicring including fully saturated, partially saturated, and unsaturatedmono-, bi, and tri-cyclic rings having 4 to 34 ring atoms, preferably, 7to 10, or 10 to 15 ring atoms having one or more heteroatoms, such as S,O, or N in each ring.

The term “sugar” refers to a mono-, di-, or tri-saccharide includingdeoxy-, thio-, and amino-saccharides. Examples of sugar include, but arenot limited to, furanose and pyranose.

The terms “halogen” and “halo” refer to radicals of fluorine, chlorine,bromine or iodine.

The term “macrolactone” refers to a large lactone ring (i.e., cyclicester) having at least 10 (e.g., 10 to 25) ring atoms.

The term “macrocyclic ether” refers to an ether having at least 10(e.g., 10 to 25) ring atoms.

The term “macrolide” refers to a chemical compound characterized by alarge lactone ring (having at least 10, e.g., 10 to 25 ring atoms)containing one or more keto and hydroxyl groups, or to any of a largegroup of antibacterial antibiotics containing a large lactone ringlinked glycosidically to one or more sugars; they are produced bycertain species of Streptomyces and inhibit protein synthesis by bindingto the 50S subunits of 70S ribosomes. Examples include erythromycin,azithromycin, and clarithromycin.

The term “ketolide” refers to a chemical compound characterized by alarge lactone ring (having at least 10 ring atoms) containing one ormore keto groups.

The term “alkyl” (or “alkenyl” or “alkynyl”) refers to a hydrocarbonchain that may be a straight chain or branched chain, containing theindicated number of carbon atoms. For example, C₁-C₁₀ indicates that thegroup may have from 1 to 10 (inclusive) carbon atoms in it. Alkenylgroups and alkynyl groups have one or more double or triplecarbon-carbon bonds, respectively, in the chain.

The term “aryl” refers to a hydrocarbon ring system (mono-cyclic orbi-cyclic) having the indicated number of carbon atoms and at least onearomatic ring. Examples of aryl moieties include, but are not limitedto, phenyl, naphthyl, and pyrenyl.

The term “heteroaryl” refers to a ring system (mono-cyclic or bi-cyclic)having the indicated number of ring atoms including carbon atoms and atleast one aromatic ring. The ring system includes at least oneheteroatom such as O, N, or S (e.g., between 1 and 4 heteroatoms,inclusive, per ring) as part of the ring system. Examples of heteroarylmoieties include, but are not limited to, pyridyl, furyl or furanyl,imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl,quinolinyl, indolyl, and thiazolyl.

The term “alkoxy” refers to an —O-alkyl radical.

The term “cycloalkyl” refers to a nonaromatic hydrocarbon ring system(mono-cyclic or bi-cyclic), containing the indicated number of carbonatoms.

The term “heterocycloalkyl” refers to a nonaromatic ring system(mono-cyclic or bi-cyclic), containing the indicated number of ringatoms including carbon atoms and at least one heteroatom such as O, N,or S (e.g., between 1 and 4 heteroatoms, inclusive, per ring) as part ofthe ring system.

“Alkyliden” is a bivalent alkyl group.

“Aryliden” is a bivalent aryl group.

“Erythrocytic cell” is a mature red blood cell that normally does nothave a nucleus: it is a very small, circular disk with both facesconcave, and contains hemoglobin, which carries oxygen to the bodytissues.

The compounds described above include the compounds themselves, as wellas their salts, if applicable. Such salts, for example, can be formedbetween a positively charged substituent (e.g., amino) on a compound andan anion. Suitable anions include, but are not limited to, chloride,bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate,trifluoroacetate, and acetate. Likewise, a negatively chargedsubstituent (e.g., carboxylate) on a compound can form a salt with acation. Suitable cations include, but are not limited to, sodium ion,potassium ion, magnesium ion, calcium ion, and an ammonium cation suchas tetramethylammonium ion.

In addition, some of the compounds of this invention have one or moredouble bonds, or one or more asymmetric centers. Such compounds canoccur as racemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms.

Further, the aforementioned compounds also include their N-oxides. Theterm “N-oxides” refers to one or more nitrogen atoms, when present in acompound, are in N-oxide form, i.e., N→O.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., treating a disease).

In another aspect, this invention features a method for treating aninflammatory disorder. The method includes administering to a subject inneed thereof an effective amount of a compound described above, whereinthe compound contains a non-antibiotic therapeutic agent that is ananti-inflammatory agent. Optionally, the method includes co-usage withother anti-inflammatory agents or therapeutic agents. The method is ableto improve therapy by concentrating a compound preferentially in immunecells including neutrophils, monocytes, eosinophils, macrophage,alveolar macrophage, B and T-lymphocytes, NK cells, giant cells, Kupfercells, glial cells, and similar target cells using a variety of means ofconcentrative compound uptake common to such cells. As such, theinvention is advantageous in that selective concentration of compoundsconforming to the definition of “therapeutic agent” above, can improvetherapy and that, for the purposes of illustration only, concentrationof agents in immune cells can confer improved characteristics oncompounds with suitable modes of action for the treatment ofinflammatory diseases.

In another aspect, the invention features a means of improving theaction of a compound in vivo by reducing its exposure to the action ofdetoxification enzymes. Such reduced exposure is a result of thestructure of the conjugate molecule causing it to be differentlyretained in the cells and organs of the organism and thus reducing orlimiting the amount of material in a given metabolic compartment.

In another aspect, the invention provides for means to improve theaction of a compound through improved retention in the cells and tissuesof the organism such that it is less efficiently eliminated by thenormal processes of circulation and filtration. Such avoidance ofelimination is, at least in part, a consequence of efficient uptake intocells resulting in reduced concentrations of the drug being availablefrom plasma.

In another aspect, the invention provides for a means of improving theaction of a drug by assisting its uptake from the intestine through theoverall effects on membrane permeability of the compound that areassociated with the invention. Uptake from oral administration is ameans of providing sustained exposure to the compound from the parts ofthe intestine to which it is permeable. Oral availability is not aproperty of all compounds.

This invention also features a method of treating a disease (e.g., aninfectious disease including viral, fungal, or parasitic diseases,cancer, allergy, metabolic, cardiovascular, pulmonary, dermatological,rheumatological or immune disease). The method comprises administeringto a subject in need thereof an effective amount of a compound describedabove, wherein the compound contains a non-antibiotic therapeutic agent(e.g., an anti-infectious agent, an anti-cancer agent, an agent fortreating a hematopoietic disorder, an agent for treating a lysosomalstorage disorder, an allergy-suppressive agent, a lipid lowering agent,a sterol synthesis modifying agent, agents active on protozoa or animmune-suppressant agent). Optionally, the method includes co-usage withother therapeutic agents. As described above, the method provides formeans to improve therapy by concentrating a compound preferentially inany of the myeloid, hepatic, respiratory, epithelial, endothelial, othertarget and immune cells. Therefore, the invention is advantageous inthat selective concentration of compounds conforming to the definitionof “therapeutic agent” above, via the methods described, can improvetherapy and that, for the purposes of illustration only, concentrationof agents in immune cells can confer improved characteristics oncompounds with suitable modes of action for the treatment of diseases ofinfectious, allergic, autoimmune, transplant, traumatic or neoplasticorigin or association.

The present invention also features a pharmaceutical compositionincluding at least one compound of this invention and a pharmaceuticallyacceptable carrier. Optionally, the pharmaceutical composition includesone or more other therapeutic agents.

This invention further features a method for making any of the compoundsdescribed above. The method includes taking any intermediate compounddelineated herein, reacting it with any one or more reagents to form acompound of this invention including any processes specificallydelineated herein.

In another aspect, this invention features a method of identifying acompound useful for enhancing efficacy of a therapeutic agent. Themethod includes incubating a compound in blood cells; separating immunecells from erythrocytic cells (e.g., by density gradient centrifugation,antibody mediated capture, lectin based capture, absorption to plastic,setting, simple centrifugation, peptide capture, activation mediatedcapture, or flow cytometry); and determining the ratio of theconcentration of the compound in the immune cells to the concentrationof the compound in the erythrocytic cells (e.g., by mass spectrometry,NMR, PET, fluorescence detection, infrared fluorescence, colorimetry,normal detection methods associated with gas chromatography, Fourriertransform spectrometry method, or radioactive detection); wherein thecompound comprises a transportophore and a therapeutic agent, in whichthe transportophore is covalently bonded to the therapeutic agent via abond or a linker. The therapeutic agent can be, for example, ananti-inflammatory agent, an anti-infectious agent, an anti-cancer agent,an allergy-suppressive agent, an immune-suppressant agent, an agent fortreating a hematopoietic disorder, a lipid lowering agent, an agent fortreating a lysosomal storage disorder, a sterol synthesis modifyingagent, agents active on protozoa, or an agent for treating a metabolicdisease.

In still further another aspect, this invention features a method fordelivering a therapeutic agent with a selective concentration. Themethod includes identifying a compound using the just-described method,and delivering the compound to a cell (e.g., a cell of respiratorytissue, a cell of neoplastic tissue, or a cell mediating allergicresponses).

Also within the scope of this invention are a composition having one ormore of the compounds of this invention (optionally including one ormore other therapeutic agents) for use in treating various diseasesdescribed above, and the use of such a composition for the manufactureof a medicament for the just-described use.

The invention provides several advantages. For example, a compound ofthis invention achieves one or more of the following improvementsrelative to a therapeutic agent itself: (i) improved uptake across theintestinal, jejunal, duodenal, colonic, or other mucosa; (ii) reducedfirst pass effect by mucosal oxygenases; (iii) reduced or altereddetoxification by degradative enzymes of the body; (iv) reduced efflux;(v) selective accumulation of the therapeutic agent in one or moreimmune, fibroblast, hepatic, renal, glial, or other target cells; (vi)potential for hydrolytic or other forms of separation on a timescalecompatible with therapy and the other desired disposition events; (vi)enhanced pharmacological effect in the target cells through greaterconcentration, sustained release, reduced substrate competition effector other mechanisms; (vii) reduced or modified dose; (viii) modifiedroute of administration; (ix) reduced or altered side effects; (x)alternative uses; and (xi) alternative formulations.

Other advantages, objects, and features of the invention will beapparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts comparison of selective uptake of diverse structure typesinto white blood cells from a complex blood mix. These data show that anamino acid (4), a macrolide (5), a sugar (1), a piperazine (2), and amacrolide (3). These data show that diverse properties can be exploitedfor concentrative uptake and that macrolides can mediate evendistribution of their cargo in the cytoplasm.

FIG. 2 depicts comparison of sugar and piperazine driven uptake of afluorophore.

FIG. 3 is bright-field overlay and fluorescent image ofpolymorphonuclear cells that have taken up a fluorescent macrolide(compound 3). The images suggest even distribution with someconcentration near the nucleus.

FIG. 4 is an example of results from a proliferation assay showingincreased efficacy of a T-L-C conjugate following concentrative uptakeinto lymphocytes.

FIG. 5 depicts a model to demonstrate the advantage of uptake intotarget cells.

FIG. 6 is an example of a response of HeLa cells to a mycophenolic acidconjugate.

FIG. 7 is an example of guanosine amelioration following treatment offresh PBMNCs with either mycophenolic acid or a T-L-C conjugate thereof.

FIG. 8 shows changes in normalized paw thickness (left) and thecorresponding arthritic scores (right) of mice treated with differentconjugates. Saline and unconjugated compounds are included as controls.

FIG. 9 shows survival of skin transplant following treatment with anexample T-L-C conjugate.

FIG. 10 shows dose tapering used in skin transplant model to study aT-L-C conjugate.

DETAILED DESCRIPTION

The invention describes a method for identifying compounds that act toimprove the uptake of therapeutic agents into cells such as those thatconstitute the immune system in mammals. The invention further comprisescompounds identified using the method and compounds that could be madebased on the teaching provided. The invention provides for the rationalimprovement of therapeutic agents intended for action in inflammatorydisease, infection, cancer, allergy, transplantation, cardiovascular,pulmonary, dermatological, rheumatological and metabolic disease. Theinvention also provides for methods to engender unoptimized molecules orthose with activity only in vitro with improved properties in vivothrough simple conjugation with molecules that meet the criteriaoutlined herein.

The method provides for the selection, in vitro, of combinations of atransportophore and a therapeutic agent that exhibits adequateconcentrative uptake and also scission with a half life adequate foragent accumulation and agent action. To identify such a combination, onecan contact a sample of native mammalian blood cells (e.g., human bloodcell), which contain at least erythrocytes, neutrophils, monocytes, andlymphocytes, with one or more transportophores and determining therelative concentration of those transportophores in the immune cells (atleast neutrophils, monocytes and lymphocytes) relative to theconcentration of them in the erythrocytes. Then, one can select atransportophore with significantly enhanced concentration in the immunecells and use the transportophore to covalently link to one or moretherapeutic agents, via a bond or a linker, to obtain a compound of thisinvention. Such a compound, containing the transportophore and thetherapeutic agent, is also concentrated in immune cells after it isincubated with blood cells. Finally, one can select a linker thatprovides appropriate cleavage rates between the transportophore and thetherapeutic agent in the target cells.

More specifically, a method described in Example 1 achieves an estimateof immune cell selective uptake in a complex and competitive biologicalfluid such that the observed uptake is relevant to the in vivo situationwhile simultaneously measuring cell specific uptake. Data from otherExamples suggest that the molecules that exhibit preferential uptake inthis system are also highly available via the oral route while alsobeing stable in the liver.

A number of variations are possible in the application of the method.The basic method includes contacting the immune cell-erythrocytepreparation with a compound or known compounds and specificallydetecting those molecules and their metabolites. A further variation isthe use of the method in screening complex mixtures of compounds withseparation and detection of the resultant cytoplasmic extracts usingMass selective detection combined with a chromatographic separationtechnique.

In a further variation, the compounds designated as transportophores areused in the synthesis of libraries such that the final reaction combineslibrary elements with a transportophore using a labile bond allowing thepreferential uptake of a compound and its likely scission in anintracellular compartment. Such libraries have the advantage that incell based assays, there is a reasonable likelihood of adequatetherapeutic agent being present at the site of action.

The compound described in the “Summary” section can be prepared bymethods known in the art, as well as by the synthetic routes disclosedherein. For example, one can react a transportophore having a reactivemoiety with a therapeutic agent having another reactive moiety. One ofthe two reactive moieties is a leaving group (e.g., —Cl, OR) and theother is a derivatizable group (e.g., —OH, or —NH—). Then, thetransportophore is covalently bonded to the therapeutic agent via areaction between the two reactive moieties. In the case when a linker ispresent, each of the two reactive moieties, independently, is a leavinggroup or a derivatizable group, and each reacts with its reactivecounterpart in the linker to form a covalent bond. Detailed routesincluding various intermediates are illustrated in the examples herein.

The chemicals used in the afore-mentioned methods may include, forexample, solvents, reagents, catalysts, protecting group anddeprotecting group reagents and the like. The methods described abovemay also additionally comprise steps, either before or after the stepsdescribed specifically herein, to add or remove suitable protectinggroups in order to ultimately allow synthesis of the compound of theformulae described herein.

As can be appreciated by the skilled artisan, the synthetic routesherein are not intended to comprise a comprehensive list of all means bywhich the compounds described and claimed in this application may besynthesized. Further methods will be evident to those of ordinary skillin the art. Additionally, the various synthetic steps described abovemay be performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995) and subsequent editions thereof.

A therapeutic agent includes any with modes of action that includeanti-inflammatory, anti-viral, anti-fungal, immune suppressant,cytostatic, anti-parasitic, lipid lowering, a sterol synthesismodifying, or metabolaregulatory action. The following is anon-exclusive list of potentially useful therapeutic agents.

Anti-Inflammatory Therapeutic Agents Non-Steroidal Anti-InflammatoryTherapeutic Agents

Diclofenac, Diflunisal, Etodolac, Fenoprofen, Floctafenine,Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen, Meclofenamate,Mefenamic, Meloxicam, Nabumetone, Naproxen, Oxaprozin, Phenylbutazone,Piroxicam, Sulindac, Tenoxicam, Tiaprofenic, Tolmetin, Acetaminophen,Aspirin, Salicylamide, acetylsalicylic acid, salicylsalicylic acid.

Celecoxib, rofecoxib, JTE-522,

Corticosteroids

Betamethasone, Budesonide, Cortisone, Dexamethasone, Hydrocortisone,Methylprednisolone, Prednisolone, Prednisone, Triamcinolone, Fluticasone

Anti-Viral Systemic:

(i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs)including but not limited to zidovudine (AZT), didanosine (ddI),zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir (ABC),emtricitabine [(−)FTC], tenofovir (PMPA) disoproxil fumarate andphosphoramidate and cyclosaligenyl pronucleotides of d4T or similarchemistries.

(ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs) includingbut not limited to, nevirapine, delavirdine, efavirenz, emivirine(MKC-442) or recent derivatives including capravirine and the novelquinoxaline, quinazolinone, phenylethylthiazolylthiourea (PETT) andemivirine (MKC-442) analogues.

(iii) protease inhibitors (PIs) including but not limited to,saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, and lopinaviror those based on alternative non-peptidic scaffolds such as cyclic urea(DMP 450), 4-hydroxy-2-pyrone (tipranavir)

(iv) viral entry, through blockade of the viral coreceptors includingbut not limited to, CXCR4 and CCR5 [bicyclams (i.e. AMD3100),polyphemusins (T22), TAK-779, MIP-1 alpha LD78 beta isoform];

(v) virus-cell fusion, through binding to the viral glycoproteinincluding but not limited to, gp41 [T-20 (pentafuside) (DP-178), T-1249(DP-107), siamycins, betulinic acid derivatives], and potentiallyzintevir, L-chicoric acid, CGP64222;

(vi) viral assembly and disassembly, through NCp7 zinc finger-targetedagents including but not limited to, [2,2′-dithiobisbenzamides (DIBAs),azadicarbonamide (ADA) and NCp7 peptide mimics];

(vii) proviral DNA integration, through integrase inhibitors such asL-chicoric acid and diketo acids (i.e. L-731,988);

(viii) viral mRNA transcription, through inhibitors of the transcription(transactivation) process (fluoroquinolone K-12, Streptomyces productEM2487, temacrazine, CGP64222).

(ix) adefovir dipivoxil, emtricitabine and entecavir, aciclovir,valaciclovir, penciclovir, famciclovir, idoxuridine, trifluridine,brivudin, ganciclovir, foscarnet, cidofovir, fomivirsen, maribavir,amantadine and rimantadine, the neuraminidase inhibitors, zanamivir andoseltamivir, ribavirin, levovirin

Antifungal, systemic—

Candicidin, Echinocandin Caspofungin,

Azole Antifungal Therapeutic Agents

-   -   Imidazoles:    -   Clotrimazole, ketoconazole, miconazole, Butoconazole, econazole,        oxiconazole, Sulconazole,

Triazoles: fluconazole, itraconazole, Terconazole, Tioconazole

Fluorinated pyrimidines, flucytosine/5-fluorocytosine, 5-fluorouracil.

Penicillium-derivatives,

griseofulvin (oral),

Allylamine and morpholine antifungal therapeutic agents, squaleneepoxidase inhibitors

naftifine, terbinafine, amorolfine,

Other,

Dapsone, Haloprogin,

Cytostatics and Immune Suppressants

Alkylating Agents,

Nitrogen Mustard Derivatives, Chlorambucil, Cyclophosphamide,Ifosfamide, Mechlorethamine, Melphalan, Uracil Mustard,

Nitrosoureas, Carmustine, Lomustine, Streptozocin, Aziridine, Thiotepa,

Methanesulfonate Ester, Busulfan, chronic myelogenous leukemia

Nonclasic Agents, Dacarbazine, Procarbazine,

Platinum Complexes, Carboplatin, Cisplatin,

Antitumor antibiotics, Dactinomycin, Daunorubicin, Doxorubicin,Idarubicin, Mitomycin, Mitoxantrone,

Antimetabolites, Fluorouracil, Floxuridine, Capecitabine,

Cytidine Analogs, Cytarabine, Gemcitabine,

Purines, Cladribine, Fludarabine, Mercaptopurine, Methotrexate,Pentostatin, Thioguanine

Plant Alkaloids, (DNA Repair Enzyme Inhibitors)

Semisynthetic Podophylline Derivatives, Etoposide, Teniposide

Taxoid Plant Alkaloids, Docetaxel, Paclitaxel,

Synthetic camptothecin

Plant Alkaloid Derivatives, Irinotecan, Topotecan,

Vinca Alkaloids, Vinblastine, Vincristine, Vinorelbine,

Other agents,

All-trans-retinoic acid, Imatinab mesylate, 2-deoxycoformycin, all-transretinoic, thalidomide calicheamycin, protein kinase inhibitors

Therapeutic Agents Active on Allergy Anti-Histamines

Astemizole, Azatadine, Brompheniramine, Cetirizine, Chlorpheniramine,Clemastine, Cyproheptadine, Dexchlorpheniramine, Dimenhydrinate,Diphenhydramine, Doxylamine, Hydroxyzine, Loratadine, Phenindamine,Terfenadine, Tripelennamine.

Lipid Lowering and Sterol Modifying Agents

Atorvastatin, Pravastatin, Simvastatin, Lovastatin, Cerivastatin,Roxuvastatin, Fluvastatin, Gemfibrozil

Also within the scope of this invention is a pharmaceutical compositionthat contains an effective amount of at least one of the compound ofthis present invention and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, mesylate, 2-naphthalenesulfonate, nicotinate, nitrate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts. Salts derived from appropriate bases include alkalimetal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammoniumand N-(alkyl)₄ ⁺ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

Further, this invention covers a method of administering an effectiveamount of one or more compounds of this invention to a subject (a human,a mammal, or an animal, e.g., dog, cat, horse, cow, or chicken) in needof treatment for a disease or disease symptom (e.g., an inflammatorydisease, an infectious disease, cancer, allergy, or an immune disease,or symptoms thereof).

The term “treating” or “treated” refers to administering a compound ofthis invention to a subject with the purpose to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve, or affect a disease, thesymptoms of the disease or the predisposition toward the disease. “Aneffective amount” refers to an amount of a compound which confers atherapeutic effect on the treated subject. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). An effective amountof the compound described above may range from about 0.1 mg/Kg to about20 mg/Kg. Effective doses will also vary, as recognized by those skilledin the art, depending on route of administration, excipient usage, andthe possibility of co-usage with other agents for treating a disease,including an inflammatory disease, a cardiovascular disease, aninfectious disease, cancer, allergy, and an immune disease.

The methods delineated herein can also include the step of identifyingthat the subject is in need of treatment of for a disorders and orcondition in a the subject. The identification can be in the judgment ofa subject or a health professional and can be subjective (e.g., opinion)or objective (e.g., measurable by a test or a diagnostic method).

The following is a non-exclusive list of diseases and disease symptoms,which may be treated or prevented by administration of the compounds andcompositions thereof herein and by the methods herein.

Inflammation and Related Disorders Inflammation Secondary to Trauma orInjury

Post traumatic regeneration injury including but not limited toIschemia, reperfusion injury, scarring, CNS trauma, spinal section,edema, repetitive strain injuries including tendonitis, carpal tunnelsyndrome,

Cardiovascular Diseases

specifically atherosclerosis, inflamed or unstable plaque associatedconditions, restinosis, infarction, thromboses, post-operativecoagulative disorders, acute stroke,

Autoimmune Diseases

Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome,Autoimmune Addison's Disease, aplastic anemia, Autoimmune HemolyticAnemia, Autoimmune Hepatitis, Behcet's Disease, biliary cirrhosis,Bullous Pemphigoid, Canavan Disease, Cardiomyopathy, CeliacSprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS),Chronic Inflammatory Demyelinating Polyneuropathy, Churg-StraussSyndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold AgglutininDisease, Crohn's Disease, dermatomyositis, Diffuse Cerebral Sclerosis ofSchilder, Discoid Lupus, Essential Mixed Cryoglobulinemia,Fibromyalgia-Fibromyositis, Fuch's heterochromic iridocyclitis, Graves'Disease, Guillain-Barré, Hashimoto's Thyroiditis, Idiopathic PulmonaryFibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy,Insulin dependent Diabetes, Intermediate uveitis, Juvenile Arthritis,Lichen Planus, Lupus, Ménière's Disease, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia Gravis, nephrotic syndrome,Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa,Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,Polymyositis and Dermatomyositis, Primary Agammag-lobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Ulcerative Colitis, Vasculitis,Vitiligo, VKH (Vogt-Koyanagi-Harada) disease, Wegener's Granulomatosis,Anti-Phospholipid Antibody Syndrome (Lupus Anticoagulant), Churg-Strauss(Allergic Granulomatosis),

Dermatomyositis/Polymyositis, Goodpasture's Syndrome, InterstitialGranulomatous Dermatitis with Arthritis, Lupus Erythematosus (SLE, DLE,SCLE), Mixed Connective Tissue Disease, Relapsing Polychondritis,HLA-B27 associated conditions including Ankylosing spondylitis,Psoriasis, Ulcerative colitis, Crohn's disease, IBD, Reiter's syndrome,Uveal diseases: Uveitis, Pediatric Uveitis, HLA-B27 Associated Uveitis,Intermediate Uveitis, Posterior Uveitis, Iritis,

Dermatological Disease

Psoriasis, atopic dermatitis, acne

Rheumatological Disease

Osteoarthritis and various forms of autoimmune arthritis.

Neurodegenerative Disease Inflammatory Degenerative Diseases

Including variants and major forms of: Alzheimer's, Huntington'sParkinson's and Creutzfeldt Jakob disease

Infection Respiratory Diseases of Diverse Origin Including:

Pharyngitis (“sore throat”), Tonsillitis, Sinusitis & Otitis Media,Influenza, Laryngo-Tracheo Bronchitis (Croup), Acute Bronchiolitis,Pneumonia, Bronchopneumonia, Bronchiolitis, Bronchitis, Acutepharyngitis with fever, Pharyngoconjunctival fever, Acute follicularconjunctivitis, Pneumonia (and pneumonitis in children), COPD, asthma,

Gastrointestinal Diseases

Gastroenteritis of diverse origin

Viral Diseases

Target viruses include but are not limited to: Paramyxo-, Picoma-,rhino-, coxsackie-, Influenza-, Herpes-, adeno-, parainfluenza-,respiratory syncytial-, echo-, corona-, Epstein-Barr-, Cytomegalo-,Varicella zoster, Hepatitis variants including hepatitis C Virus (HCV),Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis D Virus(HDV), Hepatitis E Virus (HEV), Hepatitis F Virus (HFV), Hepatitis GVirus (HGV), Human immunodeficiency-

Parasitic Diseases

Helminthiases and Similar Diseases

Larva Migrans, Toxocara canis, Hookworm Infections (Ancylostomiasis)Necator spp. Ancylostoma duodenale and Necator americanus, Filariasis,Wuchereria bancrofti & Brugia malayi, Loiasis, Ascariasis, Ascarislumbricoides-, Dracunculiasis, Schistosomiasis, Schistosoma mansoni,male & female [P Darben]-(AU), Onchocerciasis (River Blindness),Whipworm Infections, Ascaris lumbricoides and Trichuris trichiura,Trichinosis, Trichinella, Cestode Infections, Diphyllobothriasis,Diphyllobothrium spp., Echinococcosis, Echinococcisis (Hydatid Disease),Echinococcus multilocularis, Taeniasis, (Tapeworm Infection),Cysticercosis Leishmaniasis (Kala Azar), Leishmania donovani, Enterobiusvermicularis, Anal Pinworms, Dientamoebiasis, Dientamoeba fragilis,Anisakiasis, Anisakis simplex, Giardiasis, Giardia lamblia, Giardiamuris

Protozoan Infection

Acanthamoeba sp. Flagellates, Amebiasis, Naegleria, Acanthamoeba andBalamuthia, Entamoeba, Trichomonas Infections, Blastocystis hominisinfections (not on MeSH), Malaria, Plasmodium falciparum, Toxoplasmosis,Cryptosporidiosis, Cyclosporiasis, Cyclospora cayetanensis, Babesiosis,Trypanosomiasis, Trypanosomiasis, Trypanosoma brucei, Chagas Disease

Neoplastic Disease

leukemia, lymphoma, myeloma

hepatomas, other major organ carcinomas and sarcomas

glioma, neuroblastoma,

Astrocytic and glial tumors,

Invasive or non-invasive (Anaplastic (malignant) astrocytoma,Glioblastoma multiforme

variants: giant cell glioblastoma, gliosarcoma, Pilocytic astrocytoma,Subependymal giant cell astrocytoma, Pleomorphic xanthoastrocytoma)

Oligodendroglial Tumors

Ependymal cell tumors, Mixed gliomas, Neuroepithelial tumors ofuncertain origin, Tumors of the choroid plexus, Neuronal and mixedneuronal-glial tumors, Pineal Parenchyma Tumors, Tumors withneuroblastic or glioblastic elements (embryonal tumors), Neuroblastoma,ganglioneuroblastoma, Tumors of the Sellar Region, Hematopoietic tumors,Primary malignant lymphomas, Plasmacytoma, Granulocytic sarcoma, GermCell Tumors, Tumors of the Meninges

Allergy

Rhinitis, bronchitis, asthma and conditions relating to excessivelyactive or stimulated eosinophils.

Transplant Medicine

Renal, hepatic, corneal, stem cell, pulmonary, cardiac, vascular, andmyeloid transplants

Metabolic Disease,

Various disorders clustered in the liver cirrhosis, dyslipidemia,diabetes, obesity and hypercholesterolemia groupings.

BENEFITS OF THE INVENTION

The conjugates described here represent improvements on their parenttherapeutic agents in two main respects. First, these conjugates providea facile means of improving the activity of a therapeutic agent throughtheir ability to make the therapeutic agent more easily available eitherfrom the gut, or from the blood stream. This is especially important forthose therapeutic agents that have good activity in vitro but are unableto exert that activity in vivo. Where the non-manifestation of activityis related to inefficient uptake and distribution, simple conjugationsaccording to the schemes described here are an efficient means togenerate improved activity.

The invention also has specific benefits. By targeting cells, andachieving higher concentration in those cells than in plasma or generaltissue, the therapeutic agent may exert a more specific action resultingin fewer systemic side effects. Where efficacy is limited by the abilityto place sufficient therapeutic agent at the site of action, suchconcentration effects are significant in achieving improved in vitroeffect. This may be understood more clearly by examination ofnon-limiting but representative examples from different therapeuticareas.

In Examples 10-16, improved anti-inflammatory therapeutic agents aredescribed in which the active molecules are concentrated into immunecells in vitro through conjugation with a macrolide. These conjugatesdisplay superior immune suppressive and anti-inflammatory action in vivowhen compared with the effect of a mixture of the two componentmolecules in the same system. The mechanism for this action is unknownbut the effect in protection appears to be qualitatively similar for themixture and the conjugate suggesting that the conjugate is largely adelivery mechanism for the therapeutic agent. One potentialnon-exclusive explanation is that immune cells produce high levels ofarachidonate, the substrate of cyclooxygenase enzymes, resulting incompetition between this substance and NSAID therapeutic agents forsites on cyclo-oxygenase enzymes (substrate competition is known in theart as a common means of reducing the efficiency of an inhibitor).Enhanced concentration of the therapeutic agent has the potential toovercome this feedback inhibition resulting in a greater inhibition offlux through the enzyme. The conjugate also has other potential benefitsincluding the prevention of metabolism through steric effects, increasedresidence time and traffic to sites of inflammation when it is taken upinto target cells which are tropic for the inflamed tissues. Some actionof the conjugate itself cannot be ruled out when it is present at highconcentrations in a cell.

In example 24, an anti-viral therapeutic agent conjugate is cited thatalso achieves higher levels in immune cells which may act as a reservoirof integrated viral material. If therapeutic agent is selectivelyconjugated such that it is concentrated in these cells, it has twopotential benefits including, the ability to suppress viral replicationat lower systemic doses, and the ability to prevent resistance throughthe maintenance of persistently higher concentrations of therapeuticagent such that mutations with minor effect cannot accumulate.

Similar themes but contrasting mechanisms apply to the field of graftrejection where one focus of therapy is the prevention of T-cellresponses to the donor organ. Various mechanisms are known but all wouldbenefit if a greater proportion of chemical effect were focused on theT-cells themselves such that the systemic dose were reduced. Example 21.cites conjugates of mycophenolic acid that are highly concentrated inimmune cells. These conjugates are also highly bioavailable in the ratand cleave slowly to release mycophenolic acid. Despite slow cleavage,the compounds have very similar anti-proliferative activity in vitrowhen compared with unconjugated mycophenolic acid suggesting thatconcentration can compensate for slow hydrolysis such that the conjugatebecomes an intracellular reserve for the slow release of mycophenolate.

Similar advantages can be cited for cancer where those neoplasms are ofa type that takes up the conjugates to the same extent seen in immunecells. Cancers of myeloid origin are a good example of a targetneoplasm. In such cancers, concentration of the therapeutic agent haspotential to compensate for common resistance mechanisms such as geneamplification and the over expression of efflux systems. In certaincancers, the tumour is associated with an intense local inflammation.The inflammatory infiltrate may serve as a means of furtherconcentration of the conjugate drugs in the environs of the tumour.

In cardiovascular diseases such as atherosclerosis, it is commonly knownthat there is a strong inflammatory component to the events which resultin the thickening and fragmentation of the plaque. This inflammation maybe effectively reduced by the application of a range of agents includingconjugates of compounds that are anti-inflammatory in effect.

Data to support these observations may be found in various examples andis summarized here by reference in a non-limiting manner.

To practice the method of treating a disease, the compounds of thisinvention can be administered to a patient, for example, in order totreat a disease described above. The compound can, for example, beadministered in a pharmaceutically acceptable carrier such asphysiological saline, in combination with other therapeutic agents,and/or together with appropriate excipients. The compound describedherein can, for example, be administered by injection, intravenously,intraarterially, subdermally, intraperitoneally, intramuscularly, orsubcutaneously; or orally, buccally, nasally, transmucosally, topically,in an ophthalmic preparation, by inhalation, by intracranial injectionor infusion techniques, with a dosage ranging from about 0.1 to about 20mg/kg of body weight, preferably dosages between 10 mg and 1000 mg/dose,every 4 to 120 hours, or according to the requirements of the particulartherapeutic agent. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Lower or higher doses than those recited abovemay be required. Specific dosage and treatment regimens for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health status, sex, diet, time of administration, rate ofexcretion, therapeutic agent combination, the severity and course of thedisease, condition or symptoms, the patient's disposition to thedisease, condition or symptoms, and the judgment of the treatingphysician.

Pharmaceutical compositions of this invention comprise a compound ofthis invention or a pharmaceutically acceptable salt thereof; and anypharmaceutically acceptable carrier, adjuvant or vehicle. Suchcompositions may optionally comprise additional therapeutic agents. Thecompositions delineated herein include the compounds of the formulaedelineated herein, as well as additional therapeutic agents if present,in amounts effective for achieving a modulation of a disease.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying therapeutic agent delivery systems (SEDDS)such as D-alpha-tocopherol polyethyleneglycol 1000 succinate,surfactants used in pharmaceutical dosage forms such as Tweens or othersimilar polymeric delivery matrices, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein. Oil solutions or suspensionsmay also contain a long-chain alcohol diluent or dispersant, orcarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage forms suchas emulsions and or suspensions.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

A suitable in vitro assay can be used to preliminarily evaluate acompound of this invention in treating a disease. In vivo screening canalso be performed by following procedures well known in the art. See thespecific examples below.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, internet web sites,databases, patents, and patent publications.

The invention will be further described in the following example. Itshould be understood that these examples are for illustrative purposesonly and are not to be construed as limiting this invention in anymanner.

EXAMPLES

Example number Subject 1. Method for determining immune cell partition2. Transportophore: Compound 39 3. Transportophore: Compound 40 4.Transportophore: Compound 41 5. Transportophore: Compound 42 6.Transportophore: Compound 44 7. Transportophore: Compound 45 8.Transportophore: Compound 46 9. Transportophore: Compound 47 10.Transportophore: Compound 48 11. Transportophore: Compound 49 12.Transportophore: Compound 50 13. NSAID Conjugate: Diclofenac Conjugates;Compound 52, 53, 54, & 55 14. NSAID Conjugate: Compound 56 15. NSAIDConjugate: Compound 57 16. NSAID Conjugate: Compound 58 17. NSAIDConjugate: Compound 59 18. NSAID Conjugate: Compound 60 19. NSAIDConjugate: Compound 61 20. Conjugates of cytotoxic agents: Compound 6221. Conjugates of cytotoxic agents: Compound 64 22. Neotrofin conjugate:Compound 65 23. Gemfibrozil conjugate: Compound 66 24. Mycophenolic Acidconjugates: Compounds 67, 68, 69, 71, 73, 74, 75,78, 79, 80, & 81 25.Steroid Conjugates: Compounds 82, 83, 84, 85, & 86 26. StatinConjugates: Compounds 87 & 88 27. Antifungal Conjugate: Compound 89 28.Antiviral Nucleoside Conjugates: Compounds 90, 92, 94, 97, & 101 29.NSAID Conjugate: Compound 106 30. Coumarin Conjugates: Compounds 108,109 31. Imatinab Conjugate: Compound 110 32. Proliferation Assay 33.Cell-Based IMPDH Assay with Guanosine Rescue 34. Efficacy Testing ofDrugs using Collagen-Induced Arthritis in Mice 35. Efficacy Testing ofImmunosuppressive Drugs Using a Mouse Skin Transplant Model 36. Testingof Antibiotic Activity of Drugs

Example 1 Determination of the Immune Selectivity Ratio Coefficient(PISR) Uptake of Compounds

Freshly drawn heparinised blood or buffy coat preparations are used forthe determination of immune cell partition ratios. Buffy coatpreparations are preferred. These may be obtained from donor blood bysimple centrifugation of whole blood (4795 g for 10 minutes). Followingcentrifugation, plasma is collected from the surface, after which immunecells are expressed from the donor bags along with the erythrocyteslying immediately below the leukocyte layer. This ensures high yieldsand a sufficient population of erythrocytes for partition. 5 ml of theresulting cell suspension are dispensed into T25 culture flasks.Substrates are added to a final concentration between 1 and 10 μM andthe suspensions incubated at 37° C., in a 5% CO₂ atmosphere. Foranalysis of uptake kinetics, samples are withdrawn at 0, 2, 5, 10, 30,60, 90, 180, or 240 min after substrate addition. For screeningpurposes, samples are taken at 0 and 120 minutes.

Buffers and Solutions PBS 73 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8 mMNa₂HPO₄, pH 7.4 DPBS 137 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1 mM KH₂PO₄, 1mM CaCl₂, 0.5 mM MgCl₂, 5 mM Glucose, pH 7.4 Separation of Blood CellFractions—Density Gradient Centrifugation

Cell fractions were prepared using density gradient centrifugation.Mononuclear cells and polymorphonuclear cells are separated fromerythrocytes essentially by layering the cell suspension on a viscousmedium typically composed of a solution containing Ficoll or similar(commercial suppliers include: Lymphoprep, Axis Shield, 1031966;Lymphoflot HLA, 824010; or PMN Separation Medium Robbins Scientific1068-00-0). The layered suspension is then centrifuged at 600 g, 20 min,after which the cell fractions and the plasma (incubation medium)fraction are removed by gentle aspiration, washed twice in PBS buffer,followed by estimation of the cell number and pellet volume.

Analysis

Uptake of fluorescent compounds is monitored using fluorescencemicroscopy. Excitation and emission wavelengths depend of thefluorescence label in use. A typical label is a methoxy coumarin forwhich the appropriate wavelengths are 360 and 450 nm respectively.Fluorescent analogs of the compounds under study permit the estimationof appropriate uptake intervals as well as the likely intracellulardistribution of the compounds. Fluorescent analogs also allow theestimation of losses in washing or other cell manipulations.

Cell preparations are lysed in water and the debris sedimented at 16100g, 10 min. The supernatant is recovered and sub-sampled for protein andDNA content. Protein in the supernatant is precipitated by bringing thesolution to 100% v/v ethanol and centrifuging again at 16100 g, 10 min.

Compound uptake is normalized according to cytoplasmic volume of cellsin order to obtain the average concentration in the cells. Cell volumeis estimated by correlation of DNA, protein or haem content of lysedcell aliquots to cell number and packed volume prior to lysis.

Cell lysates are analysed using a HP 1100 HPLC System (AgilentTechnologies, Waldbronn, Germany) with a Kromasil 3.5μ C18, 50×2.0 mmcolumn and guard cartridge system (both, Phenomenex, Aschaffenburg,Germany) run at 30° C. A gradient elution was performed using water,0.05% formic acid (A) and acetonitrile 0.05% formic acid (B) (0 min. 5%B, 2.5 min 5% B, 2.8 min 40% B, 10.5 min 85% B, 12.0 min 95% B, 16.5 min95% B) at a flow rate of 300 μl/min. Re-equilibration of column was at5% B, at a flow rate of 750 μl/min for 2.4 min. The HPLC-eluate fromretention time 0.0 min to 2.5 min was directed directly to waste.Detection was via a UV cell at 214 nm followed by a 1/6 split to an AnAPI-qTOF 1 (Micromass, Manchester, UK) mass spectrometer, (calibrateddaily using a mixture of NaI, RbI and CsI). The mass spectrometer isroutinely operated in the positive electrospray ionization mode usingthe following settings: Capillary voltage 4000 V; cone voltage 30 V; RFLens offset 0.38 V; source block temperature 80° C.; desolvation gastemperature 140° C.; desolvation gas 240 l/h; LM/HM Resolution 0.0;Collision energy 4.0 V; Ion energy 5.0 V.

Masses are monitored according to the known or expected M/Z ratios. Ioncurrents across the expected range of masses (including metabolites) arerecorded and the chromatograms for specific masses used to estimate thepeak area for a given molecular ion (area proportional to concentrationover a given range). Normalisation to DNA and/or protein and/or haemcontent of cells (all three measured with standard methods (Bisbenzimidestaining (Sigma), BCA protein assay kit (Pierce) and haem absorbance at535 nm, respectively)) to cell number (hemocytometer count) and cellvolume is employed to calculate average compound concentration in thecell fraction (expressed in uM). Formation of metabolites or hydrolysisproducts was also monitored for each T-L-C conjugate and the rate ofhydrolysis estimated from both the total uptake and the loss ofmetabolites to the medium. The final ratio is computed by comparing theconcentration of a component in the immune cell compartment with that inboth the erythrocytes and the plasma. The P_(ISR), is then theconcentration in immune cells/concentration in erythrocytes using thesame concentration units. Thus a P_(ISR) of 2 indicates a two-foldconcentration relative to erythrocytes.

Selection and Definition of Carrier Compounds

Immune cell selectivity assays provide data in the form of micrographsof fluorescent analogs and quantitative estimates of compoundconcentration. Micrographs are useful in determining the intracellulardisposition of compounds (FIG. 1). It is apparent from the illustrationsthat compound distribution is generally uniform with some examplesappearing granular or nuclear. The analysis of fluorescent libraries bythis method provides an efficient means of selecting T molecules thatare capable of mediating the transport of diverse substances into acell. Examples of molecules assayed in this way are summarized in Table2 along with their uptake data and selectivity. These data show thatsimilar molecules with similar properties can exhibit quite differentuptake into immune cells, hence the difficulty in employing generalspecifications known in the art (Lipinski et al., 2001) Further, it isclear from the images obtained during the course of uptake (FIG. 1, FIG.2, or FIG. 3) that for some structures, the process is a slow onerelative to pure lipophilic diffusion. This is indicative of processesin uptake that depend on factors other than diffusion alone. Certaininvestigators have proposed that compounds of the macrolide type aresubject to active, protein mediated concentrative mechanisms althoughthese remain unknown (Labro, 1998). The data presented here forcompounds 4 and 5 suggest that uptake is rapid but that it varies witheach structure which does not exclude a concentrative mechanisminvolving protein action.

Compounds exhibiting high uptake are outlined in Table 2 along withsimilar structures that do not. It is clear from an inspection of thestructures that there exist a variety of chemical and physicalproperties compatible with selective entry into white blood cells. Thesedata are consistent with there being a multiplicity of mechanisms forcell entry and accumulation including passive entry and active uptake.These data further suggest that compounds with properties supposedlycompatible with facile uptake into actively metabolizing cells such asimmune cells do not exhibit such properties. Simple addition of basicfunctions is not always effective, even in in vitro screening. Incontract, addition of sugars, amino acids, or peptides can enhance entryof fluorescent compounds. Based on both the micrographs above andanalysis of immune cells following uptake, it is clear that macrolidestructures are very effective at mediating the entry of fluorescentmolecules into cells and that other basic compounds did not exhibit thisproperty. In sum, it is clear that an empirical method is the onlyreliable means of selecting and guiding synthetic chemistry towardcompounds that are well distributed and concentrated in immune cells.

TABLE 2 Compounds exhibiting concentrative uptake in white cellsConcentrative Substrate Structure cLogP uptake Compound 3

n.d. Y Compound 6

n.d. Y Compound 7

n.d. Y Compound 8

1.02 Y Compound 4

0.01 Y Compound 1

−0.79 Y Compound 9

1.86 N Compound 10

−1.14 N Compound 11

1.48 N Compound 12

−0.68 N Compound 13

−0.56 N Compound 14

−1.63 N Compound 15

n.d. Y Compound 16

n.d. Y Compound 17

n.d. Y Compound 18

n.d. Y Compound 19

n.d. N Compound 20

−1 Y Compound 21

5.95 Y Compound 22

0.84 Y Compound 23

0.89 Y Compound 24

0.58 N Compound 25

0.94 N Compound 26

1.92 Y Compound 27

2.34 Y Compound 28

1.11 Y Compound 29

1.77 N Compound 30

4.04 N Compound 31

1.83 N Compound 32

2.28 N Compound 33

1.56 N Compound 34

0.46 N Compound 35

2.88 N Compound 36

4.68 N Compound 37

3.56 N Compound 38

n.d. N

Transportophores Example 2 Compound 39

15.8 g (21.1 mmol) Azithromycin(9a-aza-9a-methyl-9-deoxo-9a-homoerythromycin A, Compound 43) wasdissolved in an icecold 6 N hydrogen chloride solution (100 ml). Thereaction mixture was stirred at 0° C. for 4 hours. The solution turnedfrom yellow to green. The solution was poured on ice (200 g) and 28 mlsodium hydroxide solution (50%) were added. The solution was extractedwith ethylacetate (300 ml). The organic layer was discarded. Afteraddition of 30 ml sodium hydroxide solution (50%) to the water layer acolorless precipitate formed. The suspension was extracted withdichloromethane (300 ml). The organic layer was separated, dried overNa₂SO₄ and concentrated under reduced pressure. After drying in highvacuum 12.8 g (100%) of a colorless foam were obtained which were usedwithout further purification.

The product was dissolved in dry dichloromethane (150 ml) and 3.1 ml(32.7 mmol) acetic acid anhydride were added. The solution was stirredat room temperature overnight, then diluted with dichloromethane (200ml) and washed with saturated sodium bicarbonate solution (150 ml). Theorganic layer was separated, dried over Na₂SO₄ and concentrated underreduced pressure. 12.3 g (92%) of compound 39 were obtained as acolorless foam, which was dried in high vacuum and used without furtherpurification.

Example 3 Compound 40

A solution of 610 mg (4.5 mmol) N-chlorosuccinimide in drydichloromethane (50 ml) was chilled to −30° C. and 0.59 ml (8 mmol)dimethylsulfide were added. A colorless precipitate formed immediatelyand the suspension was kept between −30° C. and −10C for 30 min. Thenthe reaction mixture was cooled to −40° C. and 1.9 g (3.0 mmol) ofcompound 43 were added in one portion. After 20 min the precipitate wascompletely dissolved and 0.77 ml (4.5 mmol) of ethyl diisopropylaminewere added to the colorless solution. The reaction mixture was allowedto reach ambient temperature slowly. Stirring was continued at roomtemperature for another hour. The reaction mixture was diluted withCH₂Cl₂ (50 ml) and washed with saturated sodium bicarbonate solution(100 ml). The organic layer was separated, dried over Na₂SO₄ andconcentrated under reduced pressure. A colorless oil was obtained whichwas redissolved in methanol (75 ml) and stirred at 50° C. overnight. Thesolvent was removed under reduced pressure and the residue subjected tocolumn chromatography on silica gel with chloroform/methano/7N ammoniain methanol (20:1:1) as eluent to yield 1.0 g (59%) of compound 40 as acolorless oil.

Example 4 Compound 41

To a solution of 35 ml of diethylamine in 50 ml of ethanol was added 1.5ml of 1,4-butandioldiglycidyl ether. The solution was allowed to standfor 48 h at ambient temperature. All volatiles were evaporated then andthe residue used without further purification.

Example 5 Compound 42

A solution of 15 g (20 mmol) of Compound 43 in 50 ml of acetic anhydrideis treated with 2 g of potassium carbonate and heated to reflux for 3 h.After cooling the mixture is poured onto ice and neutralized withpotassium carbonate. The mixture is extracted with ethyl acetate, washedwith water and brine and concentrated after drying (Na₂SO₄). The residueis redissolved in methanol and heated to 50° C. overnight. After removalof the methanol in vacuum the residue id redissolved in chloroform.Triethylamine (10 ml) is added and the solution cooled to 0° C. Understirring methansulfonic acid chloride (4.6 ml, 60 mmol) is added within15 min and the mixture is allowed to warm to ambient temperature. After3 h the mixture is washed with aqueous potassium carbonate solution andbrine, dried (Na₂SO₄) and concentrated in vacuum. The residue ischromatographed on silica gel, elution with ethyl acetate to yield 3.5 g(22%) of slightly yellowish foam that is used without furtherpurification.

Example 6 Compound 44

To a solution of Compound 42 (850 mg, 1 mmol) in DMF (7 mL), prepared asdescribed before, N-methyl amino-2-ethanol (0.12 ml, 2 mmol) is added.After stirring for 24 h at 70° C. the mixture is concentrated in vacuumand the residue is dissolved in ethyl acetate, washed with water andbrine, dried (Na₂SO₄) and the solvent evaporated in vacuum to yield 644mg (80%) of yellowish foam that can be used without furtherpurification.

Example 7 Compound 45

A solution of 1.1 g (1.5 mmol) of Compound 96 (See Example 24) in 5 mlof dichloromethane was combined with 415 mg (2.25 mmol) of iodo aceticacid and 450 mg (2.25 mmol) of DCC. After 2 h at ambient temperature themixture was filtered and used without purification or concentration.

Example 8 Compound 46

A solution of 2.0 g (2.5 mmol) of Compound 44 in 50 ml of 6 M HCl iskept for 15 min at ambient temperature and then extracted with 10 ml ofethyl acetate. The organic phase is discarded and the aqueous phaseneutralised with potassium carbonate and extracted with ethyl acetate.The combined organic phases are dried (Na₂SO₄) and concentrated invacuum to yield 1.47 g (91%) of a slightly yellowish solid that is usedwithout further purification.

Example 9 Compound 47

To a solution of 3.75 g (5.0 mmol) of Compound 43 in 5 ml of DMF isadded 2.5 ml of epichlorohydrin and the mixture is heated to 60-65° C.for 2 d. After cooling most of the volatiles are removed in vacuum andthe residue poured onto water and extracted with ethyl acetate. Thecombined organic phases are washed with brine, dried (Na₂SO₄) andconcentrated in vacuum. The residue is chromatographed on silica gel,elution with ethyl acetate to yield 1.4 g (40%) of a colorless waxysolid.

Example 10 Compound 48

A solution of 1.5 g (2.1 mmol) of Compound 47 and 2 ml of morpholine in15 ml of isopropanol is heated to reflux for 12 h. The mixture iscooled, poured onto water and extracted with ethyl acetate. The organicphase is washed with water, then with brine, dried (Na₂SO₄) andconcentrated in vacuum. The yellowish residue can be used withoutfurther purification, or purified by chromatography on silica gel, andeluted with chloroform/isopropanol/ammonia 20:1:1.

Example 11 Compound 49

A solution of 1.63 g (12.0 mmol) N-chlorosuccinimide in drydichloromethane (50 ml) was chilled to −40° C. and 1.3 ml (18 mmol)dimethylsulfide were added. A colorless precipitate formed immediatelyand the suspension was kept at −20° C. for 30 min. The reaction mixturewas cooled to −40° C. and 1.9 g (3.0 mmol) of compound 43 prepared asdescribed above were added in one portion. After 15 min 2.0 ml (12.0mmol) of ethyl diisopropylamine were added. The precipitate dissolvedand the solution was allowed to reach ambient temperature slowly.Stirring was continued at room temperature for 1 hour. The reactionmixture was diluted with CH₂Cl₂ (50 ml) and washed with saturated sodiumbicarbonate solution (100 ml). The organic layer was separated, driedover Na₂SO₄ and concentrated under reduced pressure. A colorless oil wasobtained which was redissolved in methanol (75 ml) and stirred at 50° C.overnight. The solvent was removed under reduced pressure and theresidue subjected to column chromatography on silica gel withchloroform/methanol/7N ammonia in methanol (30:1:1) as eluent to yield1.1 g (62%) of compound 49 as a colorless foam.

Example 12 Compound 50

To a stirred solution of 589 mg (1 mmol) of Compound 40 in methanol (20ml) was added 1.26 ml (10 mmol) of hydrogen peroxide (30%). Afterstirring for 3 days at room temperature the reaction mixture was chilledto −78° C. and a solution of 1.26 g (10 mmol) sodium sulfite in 10 ml ofwater was added. The suspension was allowed to warm up to roomtemperature and then all volatile compounds removed under reducedpressure. The residue was resuspended in methanol and filtered. Thefiltrate was concentrated under reduced pressure to furnish the crudeproduct. Column chromatography on silica gel with chloroform/methanol/7Nammonia in methanol (15:4:1) as the eluent yielded 327 mg (54%) ofcompound 51 as a colorless oil.

To a stirred solution of 870 mg (1.4 mmol) of Compound 51 in dryN,N′-dimethylacetamide (20 ml) was added 370 mg (3.3 mmol) potassiumtert-butoxide. The colorless solution turned slowly orange and waschilled to −15° C. 0.25 ml (2.2 mmol) ethyl bromoacetate were added andthe reaction mixture allowed to warm up to room temperature. 2.0 ml oftriethylamine were added and stirring continued for another hour. Thereaction mixture was diluted with ethanol (20 ml) and acetic acid (2.0ml) and 0.3 g of Pd/C (10%) were added. The reaction mixture was shakenunder an atmosphere of hydrogen overnight. After filtration all volatilecompounds were removed under reduced pressure. The crude product wassubjected to column chromatography on silica gel withchloroform/methanol/7N ammonia in methanol (15:1:1) as the eluent toyield 340 mg (35%) of compound 50 as a colorless oil.

Acids Example 13 Diclofenac Conjugates Compound 52

A solution of Diclofenac (0.67 g; 2.25 mmol) in methylene chloride (10ml), is treated with N,N′-carbonyldiimidazole (0.38 g; 2.25 mmol). Afterstirring for 30 min at RT, Compound 43 (0.57 g; 0.75 mmol) is added.Reaction is stirred for 3 h at RT. The reaction solution wasconcentrated in vacuum and the residue purified by column chromatographyon silica gel, elution with chloroform/isopropanol/methanolic ammonia60:1:1 to yield Compound 52 (0.15 g; yield: 20%) as a white foam.

Compound 53

A suspension of 590 mg (2.0 mmol) of diclofenac in 6 ml ofdichloromethane is treated with 324 mg (2.0 mmol) of carbonyldiimidazole at 0° C. After 5 min at this temperature 294 mg (0.5 mmol)of Compound 40 is added and the mixture kept at ambient temperature for48 h. The mixture is then concentrated and the residue chromatographedon silica gel, elution with chloroform/isopropanol/methanolic ammonia40:1:1 to yield 330 mg (76%) of a colorless solid.

Compound 54

To a turbid solution of 740 mg (2.5 mmol) diclofenac in drydichloromethane (20 ml) was added a solution of 1N hydrogen chloride inether (2.5 ml) and 440 mg (2.7 mmol) of 1,1′-carbonyldiimidazole. Thesolution was stirred for 60 min at room temperature. Then 587 mg (1mmol) of Compound 49 were added and stirring continued overnight. Themixture was diluted with CH₂Cl₂ (30 ml) and washed with saturated sodiumbicarbonate solution (50 ml). The organic layer was separated, driedover Na₂SO₄ and concentrated under reduced pressure to furnish a reddishoil. Column chromatography on silica gel with chloroform/methanol/7Nammonia in methanol (30:1:1) as eluent yielded 450 mg (52%) of compound54 as a colorless oil.

Compound 55

To a turbid solution of 740 mg (2.5 mmol) diclofenac in drydichloromethane (20 ml) was added a solution of 1N hydrogen chloride inether (2.5 ml) and 440 mg (2.7 mmol) of 1,1′-carbonyldiimidazole. Thesolution was stirred for 60 min at room temperature. Then 340 mg (0.5mmol) of compound 50 prepared as described above were added and stirringcontinued overnight. The mixture was diluted with CH₂Cl₂ (30 ml) andwashed with saturated sodium bicarbonate solution (50 ml). The organiclayer was separated, dried over Na₂SO₄ and concentrated under reducedpressure to furnish a reddish oil. Column chromatography on silica gelwith chloroform/methanol/7N ammonia in methanol (10:1:1) as eluentyielded 214 mg (45%) of compound 55 as a colorless oil.

Example 14 Compound 56

A solution of Meclofenamic acid (0.36 g; 1.2 mmol) in methylene chloride(15 ml), is treated with N,N′-carbonyldiimidazole (0.20 g; 1.2 mmol).After stirring for 30 min at RT, Compound 44 (0.47 g; 0.75 mmol) isadded. Reaction is stirred for 3 h at RT. The reaction solution isconcentrated in vacuum and the residue purified by column chromatographyon silica gel, elution with chloroform/isopropanol/methanolic ammonia60:1:1. The appropriate fractions are collected and concentrated toyield 0.17 g (25%) of Compound 56 as a white foam.

Example 15 Compound 57

8.

A solution of Mefenamic acid (0.29 g; 1.2 mmol) in methylene chloride (5ml), is treated with N,N′-carbonyldiimidazole (0.20 g; 1.2 mmol). Afterstirring for 30 min at ambient temperature, Compound 44 (0.47 g; 0.75mmol) is added. Reaction is stirred for 3 h at ambient temperature. Thereaction solution is concentrated in vacuum and the residue purified bycolumn chromatography, elution with chloroform/isopropanol/methanolicammonia 60:1:1. The appropriate fractions are collected and concentratedto produce Compound 57 (0.16 g; yield: 25%) as a white foam.

Example 16 Compound 58

9.

A solution of Indomethacin (0.80 g; 2.25 mmol) in methylene chloride (10ml), is treated with N,N′-carbonyldiimidazole (0.38 g, 2.25 mmol). Afterstirring for 30 min at RT, Compound 40 (0.44 g; 0.75 mmol) is added.Reaction is stirred for 3 h at RT. The reaction solution wasconcentrated in vacuum and the residue purified by column chromatographyon silica gel, elution with isopropanol to yield 0.20 g (25%) ofCompound 58 a white foam.

Example 17 Compound 59

A solution of 360 mg (2.0 mmol) of acetyl salicylic acid is treated with1.5 ml (16 mmol) oxalylic chloride in 10 ml of chloroform. A drop of DMFis added and the mixture is allowed to stand at ambient temperature for1 h. All volatiles are removed in vacuum and the residue dissolved in 20ml of dichloromethane. After cooling to 0° C. 376 mg (0.65 mmol) ofCompound 104 (See Example 24) is added followed by 1 ml of pyridine. Themixture is allowed to warm to ambient temperature and after 2 hconcentrated in vacuum. The residue is chromatographed on silica gel,elution with chloroform/isopropanol/methanolic ammonia 60:1:1 to yield205 mg (35%) of Compound 59 as a white solid.

Example 18 Compound 60

A solution of Ibuprofen (0.47 g; 2.25 mmol) in methylene chloride (10ml), is treated with N,N′-carbonyldiimidazole (0.38 g, 2.25 mmol). Afterstirring for 30 min. at RT, Compound 43 (0.56 g; 0.75 mmol) is added.Reaction is stirred for 3 h at RT. The reaction solution wasconcentrated in vacuum and the residue purified by column chromatographyon silica gel, elution with isopropanol to yield 0.17 g (25%) of whitefoam, Compound 60.

Example 19 Compound 61

10.

A solution of flurbiprofen (0.27 g; 1.2 mmol) in methylene chloride (5ml), is treated with N,N′-carbonyldiimidazole (0.20 g; 1.2 mmol). Afterstirring for 30 min. at ambient temperature, Compound 46 (0.47 g; 0.75mmol) is added. Reaction is stirred for 3 h at ambient temperature. Thereaction solution was concentrated in vacuum and the residue purified bycolumn chromatography on silica gel, and elution withchloroform/isopropanol/methanolic ammonia 60:1:1 to yield 0.16 g (25%)of product Compound 61, a white foam.

Example 20 Compound 62

600 mg of melphalan (63) is suspended in 25 ml of water containing 500mg of sodium carbonate. 10 ml of dioxane is added and 1 ml of aceticanhydride. After stirring at ambient temperature for 1 h citric acid isadded and the mixture extracted with ethyl acetate. After washing withwater and brine the organic phase is dried (sodium sulfate) andconcentrated in vacuum. Removal of all volatiles yields the crudeN-acetylmelphalan that is carried on to the next step without furtherpurification.

A solution of N-acetylmelphalan (0.35 g; 1.0 mmol) dissolved inmethylene chloride (5 ml), is treated with N,N′-carbonyldiimidazole(0.17 g; 1.0 mmol). After stirring for 30 min. at RT, Compound 43 (0.29g; 0.50 mmol) is added. After 3 h the reaction solution is concentratedin vacuum and the residue purified by column chromatography on silicagel, elution with chloroform/isopropanol/methanolic ammonia 60:1:1. Theappropriate fractions are collected and concentrated to produce 0.12 g(25%) of Compound 62 as a white foam.

Example 21 Compound 64

To a solution of chlorambucil (303 mg; 1 mmol) in methylene chloride (5ml), is added N,N′-carbonyldiimidazole (130 mg; 1 mmol). After 30 minstirring at ambient temperature, Compound 43 (750 mg; 1 mmol) is added.After stirring at the same temperature for 3 h the mixture is washedwith ice water and ice cold Na₂CO₃ solution. The organic layer is dried(Na2SO4), concentrated in vacuum and chromatographed on silica gel,elution with isopropanol to afford 207 mg (20%) of a white foam,Compound 64, MS (M+2H⁺: 517).

Example 22 Compound 65

A suspension of neotrofin (0.73 g; 2.25 mmol) in methylene chloride (15ml), is treated with N,N′-carbonyldiimidazole (0.38 g; 2.25 mmol). Afterstirring for 2 h at ambient temperature Compound 43 (0.57 g; 0.75 mmol)is added. Reaction is stirred for 48 h at ambient temperature. Thereaction solution is concentrated in vacuum and the residue purified bycolumn chromatography on silica gel, elution withchloroform/isopropanol/methanolic ammonia 60:1:1. The appropriatefractions are collected and concentrated to produce Compound 65 (0.20 g;yield: 25%) as a white foam.

Example 23 Compound 66

A solution of Gemfibrozil (0.56 g; 2.25 mmol) in methylene chloride (10ml), is treated with N,N′-carbonyldiimidazole (0.38 g; 2.25 mmol). Afterstirring for 30 min. at ambient temperature, Compound 40 (0.44 g; 0.75mmol) is added. Reaction is stirred for 48 h at ambient temperature. Thereaction solution was concentrated in vacuum and the residue purified bycolumn chromatography on silica gel, elution withchloroform/isopropanol/methanolic ammonia 60:1:1. The appropriatefractions are collected and concentrated to produce Compound 66 (0.15 g;yield: 25%) as a white foam.

Example 24 Mycophenolate Derivatives Compound 67

To a mixture of 375 mg of Compound 43, 400 mg of triphenyl phosphine and960 mg of mycophenolic acid is added 4 ml of THF under nitrogen.Diisopropyl azodicarboxylate (0.3 ml) is added drop wise at 0° C. within4 h while the reaction mixture is rapidly stirred. Cooling is continuedfor another 4 h and the mixture then allowed to warm to ambienttemperature within 5 h. The mixture is then dissolved in a mixtureconsisting of 50 ml of toluene and 20 ml of ethyl acetate and extractedwith ice-cold 0.5 M hydrogen chloride (3×150 ml). The aqueous phase iswashed several times with small amounts of toluene and then withpotassium carbonate till no foaming occurs any more upon addition. Themixture is extracted with dichloromethane and the organic phase iswashed with brine, dried and concentrated in vacuum to yield white solidfoam that can be used without further purification, or further purifiedon a silica gel column, eluting with isopropanol.

Compound 68

To a solution of 170 mg of Compound 41, 400 mg of triphenylphosphine and500 mg of mycophenolic acid in 3 ml of THF were added under nitrogen 0.3ml of diisopropyl azodicarboxylate within 4 h at 0°. The mixture wasallowed to stir at 0° C. for 3 h and was then allowed to warm to ambienttemperature slowly. The reaction mixture was diluted with 70 ml oftoluene and 30 ml of ethyl acetate and extracted repeatedly withice-cold 0.5 M hydrogen chloride. The combined aqueous phases wereextracted several times with a small quantity of toluene. The organicphases were discarded. The aqueous phase was treated with potassiumcarbonate till gas evolution had stopped and was then extracted withdichloromethane. Drying (sodium sulfate) and concentration in vacuumyielded an oily residue that was purified by filtration through a shortpad of silica gel (elution with ethyl acetate-triethylamine) to afford185 mg (39%).

Compound 69

A solution of 750 mg (1.0 mmol) of Compound 43 in 10 ml ofdichloromethane is treated with 100 mg (1.0 mmol) of succinic anhydride.After stirring at ambient temperature for 12 h the mixture isconcentrated in vacuum to yield Compound 70, a colorless solid that isused with out further purification.

To a solution of mycophenolic acid ethyl ester (175 mg, 0.5 mmol) inchloroform (1 ml) is added ethyldiisopropylamine (85 μL, 0.5 mmol).After stirring for 1 min Compound 70 (425 mg, 0.5 mmol) is added undernitrogen at 0-4° C. and afterwards chlor-N,N,2-trimethylpropenamine (1mL; 0.5 mmol; 0.5 mmol/mL solution in chloroform) is added drop-wise.The mixture is stirred at 0-4° C. for 0.5 h and 12 h at roomtemperature. The mixture is concentrated in vacuum and the residuechromatographed on silica gel, elution withchloroform/2-propanol/ammonia 30:1:1, affording 147 mg (25%) of acolorless solid.

Compound 71

To mycophenolic acid (0.50 g; 1.5 mmol) and carbonyldiimidazole (0.25 g;1.5 mmol), dissolved in methylene chloride (2 mL) is added after 1minute stirring at 0-4° C. a solution of Compound 72 (0.27 g, 0.5 mmol)in 1 ml of dichloromethane. After stirring for 30 min at 0-4° C. themixture is stirred for 12 h at room temperature. The mixture isconcentrated in vacuum and the residue chromatographed on silica gel,elution with chloroform/2-propanol/ammonia 30:1:1, affording 98 mg (23%)of a colorless solid.

Compound 73

Mycophenolic acid (0.50 g; 1.5 mmol) is suspended in 3 ml ofdichloromethane and treated with carbonyldiimidazole (0.25 g; 1.5 mmol).After 10 min a solution of Compound 44 (0.38 g; 0.5 mmol) indichloromethane is added. After 30 min at 0° C. the mixture is stirredfor 12 h at room temperature. The mixture is concentrated in vacuum andthe residue chromatographed on silica gel, elution withchloroform/2-propanol/ammonia 30:1:1, affording 126 mg (22%) of acolorless foam.

Compound 74

A solution of Compound 73 in 6 M HCl (20 ml) is kept at ambienttemperature for 15 min and then extracted S ml of ethyl acetate. Theorganic phase is discarded and the aqueous phase neutralized withpotassium carbonate and extracted with methylene chloride. The organicphase is dried (Na2SO4) and concentrated in vacuum to yield 400 mg (84%)of a colorless foam.

Compound 75

A solution of 1.1 g (1.5 mmol) of Compound 43 in 5 ml of dichloromethanewas combined with 415 mg (2.25 mmol) of iodoacetic acid and 450 mg (2.25mmol) of DCC. After 2 h at ambient temperature the mixture was filteredand the resulting Compound 76 was used without purification orconcentration.

A solution 720 mg of (0.8 mmol) of Compound 76 in 3 ml ofdichloromethane, prepared as described above, is diluted with 20 ml ofDMF and 0.1 ml (1.2 mmol) of N-methyl amino ethanol is added. Themixture is kept at ambient temperature for 24 h. The mixture is pouredonto a solution of potassium carbonate in water and extracted withdichloromethane. The organic phase is washed with brine, dried (Na2SO4and concentrated in vacuum. The residue is chromatographed on silicagel, elution with chloroform/isopropanol/methanolic ammonia 80:1:1 toyield 210 mg (31%) of Compound 77, a colorless solid.

A suspension of mycophenolic acid (0.50 g; 1.5 mmol) in 8 ml ofdichloromethane was treated with carbonyldiimidazole (0.25 g; 1.5 mmolat0° C. After 10 min a solution of Compound 77 (0.40 g, 0.5 mmol) in 2 mlof dichloromethane was added. After stirring for 30 min. at 0-4° C. themixture is stirred for 24 h at room temperature. The mixture isconcentrated in vacuum and the residue chromatographed on silica gel,elution with chloroform/2-propanol/ammonia 30:1:1, affording 175 mg(32%) of Compound 75.

Compound 78

A solution of Compound 75 (550 mg, 0.5 mmol) prepared as describedbefore in 20 ml of 6 M HCl is kept at ambient temperature for 10 min andthen extracted with diethylether. The organic phase is discarded and theaqueous phase neutralized with potassium carbonate and extracted withdichloromethane. The organic phase is washed with brine, dried (Na₂SO₄)and concentrated in vacuum to yield 420 mg (89%) as a slightly yellowishfoam.

Compound 79

A suspension of mycophenolic acid (0.30 g; 0.9 mmol) in 8 ml ofdichloromethane is treated with carbonyldiimidazole (0.15 g; 0.9 mmol)at 0° C. After 10 min a solution of Compound 40 (0.20 g, 0.3 mmol) in 2ml of dichloromethane was added. After stirring for 30 min. at 0-4° C.the mixture is stirred for 24 h at room temperature. The mixture isconcentrated in vacuum and the residue chromatographed on silica gel,elution with chloroform/2-propanol/ammonia 30:1:1, affording 100 mg(35%) of a colorless foam.

Compound 80

11.

A mixture of 120 mg of Compound 48, 320 mg of mycophenolic acid and 300mg of triphenyl phosphine is dissolved in 2 ml of THF under nitrogen. At0° C. 0.1 ml (0.5 mmol) diisopropyl azodicarboxylate is added in severalportions within 4 h. After this time the mixture is allowed to warm toambient temperature overnight. The reaction mixture is concentrated invacuum and chromatographed on silica gel, elution with isopropanol.

Compound 81

To a solution of 188 mg of Compound 41, 400 mg of triphenylphosphine and500 mg of mycophenolic acid in 3 ml of THF were added under nitrogen 0.3ml of diisopropyl azodicarboxylate within 4 h at 0° C. The mixture wasallowed to stir at 0° C. for 3 h and was then allowed to warm to ambienttemperature slowly. The reaction mixture was diluted with 70 ml oftoluene and 30 ml of ethyl acetate and extracted repeatedly withice-cold 0.5 M hydrogen chloride. The combined aqueous phases wereextracted several times with a small quantity of toluene. The organicphases were discarded. The aqueous phase was treated with potassiumcarbonate until gas evolution had stopped and was then extracted withdichloromethane. Drying (Na₂SO₄) and concentration in vacuum yielded anoily residue that was purified by filtration through a short pad ofsilica gel (elution with ethyl acetate-triethyl amin) to yield 255 mg(52%) of a yellowish oil.

Example 25 Steroid Conjugates Compound 82

Prednisolone (180 mg, 0.5 mmol) is suspended in 3 ml of chloroform and55 mg (0.55 mmol) of succinic anhydride is added. After 24 h at ambienttemperature the mixture is cooled to 0° C. and 325 mg of Compound 46(0.5 mmol) is added followed by chlor-N,N,2-trimethylpropenamine (0.2ml, 1.5 mmol) in several portions. The resulting solution is subjectedto column chromatography on silica gel, elution with isopropanol toyield a white solid.

Compound 83

Dexamethasone (196 mg, 0.5 mmol) is suspended in 3 ml of chloroform and55 mg (0.55 mmol) of succinic anhydride is added. After 24 h at ambienttemperature 375 mg of Compound 43 (0.5 mmol) is added followed bychloro-N,N,2-trimethylpropenamine (0.2 ml, 1.5 mmol) in severalportions. The resulting solution is after 1 h subjected to columnchromatography on silica gel, elution with isopropanol to yield 198 mg(32%) of a white solid.

Compound 84

A solution of 295 mg (0.5 mmol) of Compound 40 in 4 ml ofdichloromethane is treated with 55 mg (0.55 mmol) of succinic anhydrideand the mixture stirred at ambient temperature overnight. To thereaction mixture diethylstilbestrol (174 mg, 0.5 mmol) and 0.15 ml ofdiisopropylethylamine is added followed by 0.133 ml ofchloro-N,N,2-trimethylpropenamine (1.0 mmol) in several portions. After1 h the reaction mixture is concentrated in vacuum and the residuechromatographed on silica gel, elution with ethyl acetate, changing toisopropanol, to yield 74 mg (16%) of a colorless solid.

Compound 85

12.

A solution of 217 mg (0.5 mmol) of triamcinolone acetonide, 55 mg (0.55mmol) of succinic anhydride in 3 ml of dichloromthane and 1 ml ofpyridine is reacted 2 d at ambient temperature. After this period allvolatiles are removed and the residue taken up in THF. To this mixture100 mg (0.62 mmol) of carbonyldiimidazole is added under nitrogen,followed by 300 mg (0.51 mmol) of Compound 40. The mixture was heated to50° C. for 36 h. After cooling the mixture was concentrated in vacuumand the residue chromatographed on silica gel, elution with ethylacetate, changing to isopropanol to yield 34 mg (6%) of a colorlesssolid.

Compound 86

13.

Prednisolone (180 mg, 0.5 mmol) is suspended in 3 ml of chloroform and55 mg (0.55 mmol) of succinic anhydride is added. After 24 h at ambienttemperature the mixture is cooled to 0° C. and 295 mg of Compound 40(0.5 mmol) is added followed by chlor-N,N,2-trimethylpropenamine (0.2ml, 1.5 mmol) in several portions. The resulting solution is subjectedto column chromatography on silica gel, elution with isopropanol toyield 165 mg (32%) of a white solid

Example 26 Statins Compound 87

A solution of 560 mg (1 mmol) of atorvastatin in 10 ml ofdichloromethane is treated with 2 ml of a 1 M solution of HCl in diethylether at ambient temperature for 12 h. The reaction mixture is washedwith water and brine, dried (Na₂SO₄) and concentrated in vacuum. Theresidue is dissolved in 8 ml of chloroform and treated with 120 mg (1.2mmol) of succinic anhydride and 123 mg (1.0 mmol) of DMAP undernitrogen. After 24 h at ambient temperature 194 mg (1.2 mmol) ofcarbonyldiimidazole is added, followed after 10 min by 700 mg (1.2 mmol)of Compound 40. The mixture is heated to 50° C. for 36 h and thencooled, concentrated in vacuum and chromatographed on silica gel,elution with chloroform/2-propanol/ammonia 30:1:1 to yield 125 mg (10%)of a colorless solid.

Compound 88

A solution of 25 mg (0.06 mmol) of lovastatin in 1 ml of dichloromethanewas treated with 10 mg (0.1 mmol) of succinic anhydride under nitrogen.The mixture was kept at ambient temperature for 48 h and then 12 mg ofcarbonyldiimidazole is added followed after 10 min by 70 mg (0.11 mmol)of Compound 46. After stirring for 48 h at ambient temperature themixture is concentrated in vacuum and the residue chromatographed onsilica gel, elution with chloroform/2-propanol/ammonia 30:1:1 to yield13 mg (19%) a white solid.

Example 27 Antifungal Conjugate Compound 89

To a stirred solution of Fluconazole (0.67 g, 2.2 mmol) in anhydrousCH₂Cl₂ (20 ml) was added triethylamine (0.31 ml, 2.2 mmol) and succinicanhydride (0.22 g, 2.2 mmol). After stirring for 2 hours at ambienttemperature N,N′-carbonyldiimidazole (0.37 g, 2.3 mmol) was added andstirred for another 2 hours. Subsequently Compound 43 (1.12 g, 1.5 mmol)was added and stirring continued overnight. Then the reaction mixturewas diluted with CH₂Cl₂ (20 ml) and a saturated aqueous solution ofsodium bicarbonate (30 ml). After separation, the organic layer wasdried over Na₂SO₄, filtered and then concentrated under reduced pressureto furnish the crude product. Silica gel chromatography withTHF-Hexane-NEt₃ (10:10:0.1) yielded Compound 89 as a white solid (0.20g, 12%).

Alcohols Example 28 Nucleosides Compound 90

To a mixture of 800 mg glutaric acid (6 mmol, 6 eq.) and 500 mg CDI (3mmol, 3 eq.) dissolved in 10 ml dry acetonitrile and stirred for 30minutes at room temperature under argon, is added a solution of 750 mgCompound 43 (1 mmol) in the presence of a catalytic amount of DMAPdissolved in 5 ml acetonitrile. The reaction is refluxed overnight.

The solvent is removed in vacuo. The crude mixture is then purified bychromatography with chloroform/methanol/ammonia (94.5:10:0.5). Thecollected fractions yielded a white solid (340 mg, 45%). The expectedCompound 91 is characterized by TLC (R_(f)=0.4 inchloroform/methanol/ammonia (90:9:1)) and by MS ([M+H]+=863).

43 mg Compound 91 (0.05 mmol) and 15 mg Abacavir (0.05 mmol) are reactedin the presence of 12 mg DCC (0.06 mmol, 1.2 eq.). The mixture isdissolved in 1 ml of dry THF and stirred overnight at room temperature.The cloudy solution is filtered off and the solvent is removed in vacuo.The crude product is purified by chromatography. The collected fractionsare concentrated to yield a white solid (20 mg, 40%). The expectedCompound 90 is characterized by TLC (R_(f)=0.6 inchloroform/methanol/ammonia (90:9:1)) and by MS (M+2H, 566).

This protocol can be applied to other alcohols, some of which are listedin Table 3

TABLE 3 Representative class of alcohol compounds, which can be used inconjugation reactions. Pencyclovir

Zalcitabine

Lamivudine

Gemcitabine

Carbovir

Cytarabine

Abacavir

Levovirin-Benzylidene acetal

Lodenosine

Ribavirin-Benzyliden acetal

MercaptopurineRiboside-benzylidenacetal

Compound 92

200 mg of Compound 43 (0.27 mmol) are treated with 30 mg succinicanhydride (0.3 mmol, 1.1 eq.) in 1 ml pyridine in the presence of acatalytic amount of DMAP. The reaction is stirred for 5 h at 40° C.After the completion of the reaction the product is separated byprecipitation using hexane. The solution is decanted, and the recoveredprecipitate is washed several times with hexane to remove pyridine. Theisolated compound is dried by high vacuum and yielded to a white solid(180 mg, 90%). The expected Compound 93 is characterized by MS([M+H]+=850).

42 mg of Compound 93 (0.05 mmol) and 13 mg AZT (0.05 mmol) are coupledby using 11 mg DCC (0.055 mmol) in 0.5 ml of dry THF. The mixture isstirred overnight at room temperature. The cloudy solution is thenfiltered off to remove the urea.

The isolated crude product, obtained after removal of solvent, ispurified by chromatography. The collected fractions yield afterevaporation to a white solid (30 mg, 50%). The expected compoundCompound 92 is characterized by TLC (R_(f)=0.3 inchloroform/methanol/ammonia (90:9:1)) and by MS (M+2H, 549.7).

This protocol can be applied to other alcohols, some of which are listedin Table 3.

Compound 94

To a cloudy solution of 52 mg AZT (0.2 mmol), 43 mg 5-bromovaleric acid(0.24 mmol, 1.2 eq.), 106 mg BOP (0.24 mmol, 1.2 eq.), and a catalyticamount of DMAP in 1 ml dry THF are added 100 μl triethylamine (72 mg,700 μmol, 3 eq.). The clear solution is then stirred for 4 h at roomtemperature. After completion of reaction the crude mixture is purifiedby preparative TLC. Removal from the plate yields a yellowish oily solid(60 mg, 70%). The expected compound 95 is characterized by TLC(R_(f)=0.7, chloroform/methanol/ammonia 90:9:1).

A solution of 6.0 g (8.0 mmol) of Compound 43 in 20 ml of THF is treatedwith 1.97 g (8.8 mmol) of N-iodosuccinic imide in several portions at 0°C. The mixture is kept at 10° C. for 12 h and then poured into asolution of potassium carbonate in water and extracted withdichloromethane. The organic phase is dried (Na2SO4), concentrated invacuum and the residue chromatographed on silica gel, elution withcyclohexane/ethyl acetate/isopropanol/triethylamin 9:1:0.2:0.2 to yield1.5 g (34%) of Compound 96, a colorless solid.

To a cloudy solution of 8 mg Compound 95 (0.02 mmol) and 26 mg Compound96 (0.035 mmol, 2 eq.) in acetonitrile (0.5 ml) is added am excess ofpotassium carbonate. The reaction mixture is then set to 50° C. for 48h. The crude mixture is purified by chromatography to yield a yellowishsolid (4.5 mg, 20%). The expected Compound 94 is characterized by TLC(R_(f)=0.5 in chloroform/methanol/ammonia (90:9:1)) and by MS([M+H]+=1113).

This protocol can be applied to other alcohols, some of which are listedin Table 3.

Compound 97

665 mg benzylidene-protected Ribavirin (2 mmol), 1.34 g glutaric acid(10 mmol, 5 eq.), 1 g CDI (6.2 mmol), and a catalytic amount of DMAP aresuspended and heated in 20 ml of Chloroform for 3 h. The solvent isremoved and the residue suspended in 1 M HCl, saturated with sodiumchloride. The mixture is extracted twice with ethyl acetate, the organiclayers are dried over sodium sulphate and evaporated to dryness. Theresidue is purified by chromatography to yield 760 mg (85%) of Compound98, characterized by TLC (R_(f)=0.16 in THF/Hexane/Acetic acid 7:7:0.5)and MS ([M+H]+=447).

To a solution of 267 mg Z-β-alanine (1.2 mmol, 1.2 eq.) and 190 mg CDI(1.2 mmol, 1.2 eq.) in 2 ml of dry THF, which had been stirred for 30minutes at room temperature under argon, 749 mg Compound 43 are added (1mmol). The mixture is then stirred overnight at 40° C. The clear andcolorless solution is purified by flash chromatography. The collectedfractions are concentrated to yield to a yellow solid (460 mg, 50%). Theexpected compound is characterized by TLC (R_(f)=0.2 inchloroform/methanol/ammonia (90:9:1)) and by MS ([M+H]+=954.7). 450 mgof this compound (0.45 mmol) are dissolved in 5 ml of ethanol, to whichan excess of Pd/C is added under argon. The flask with hydrogen. Themixture is shaken gently overnight at room temperature. The Pd/C isremoved passing the solution through a celite plug. The removal ofsolvent yielded a slightly black solid (280 mg, 76%), a mixture ofCompound 99 and Compound 43. The expected Compound 99 is characterizedby TLC (R_(f)=0.2 in chloroform/methanol/ammonia (94.5:5:0.5)) and by MS([M+H]+=890.5).

To a cloudy solution of 23 mg Compound 98 (0.05 mmol), and 41 mg freeCompound 99. (0.05 mmol), 25 mg BOP (0.055 mmol, 1.1 eq.) and acatalytic amount of DMAP in 0.5 ml of dry THF are added 15 μl oftriethylamine (11 mg, 0.11 mmol, 2 eq.). The clear solution is stirredovernight at room temperature. The mixture is purified by chromatographyand yields 5 mg (10%) of a light yellowish solid. The expected Compound100 is characterized by TLC (R_(f)=0.25 in chloroform/methanol/ammonia(90:9:1)) and by MS ([M+H]+=1249).

5 mg of Compound 100 are dissolved in 5 ml 2-propanol and a tip of aspatula of Pd/C is added. The mixture is hydrogenated overnight. Thecatalyst is extracted with ethyl acetate and the extract purified bypreparative TLC to yield 2.3 mg of the desired Compound 97,characterized by TLC (R_(f)=0.45,chlororform/2-propanol/methanol/ammonia 25:3:1:1) and MS ([M+H]+=1161).

Compound 101

900 mg of Compound 43 (1.2 mmol) are treated with 10 ml 12 N HCl in aniced-water bath overnight. The completed reaction is worked up by anextraction with chloroform. The aqueous phase is further neutralized at0° C. by addition of potassium hydroxide pellets to have a pH at 9-10.The orange basic aqueous phase is then extracted several times withchloroform. The combined organic layer is washed with brine and thendried over sodium sulphate. The crude product after evaporation of thesolvent is purified by flash column chromatography to yield to a lightyellowish solid (400 mg, 90%). The expected Compound 102 ischaracterized by TLC (R_(f)=0.35 in chloroform/methanol/ammonia(90:9:1)) and by MS ([M+H]+=434).

300 mg of the Compound 102 (0.7 mmol), 190 mg iodoacetic acid (1 mmol,1.5 eq.) and 210 mg DCC (1 mmol, 1.5 eq.) are dissolved in 5 ml of drychloroform at 0° C. under argon atmosphere under protection from light.The mixture is stirred overnight at room temperature. The yellowishcloudy solution is filtered off and the filtrate is concentrated undervacuum. Chromatography yields a fraction that contains mainly themonoacetylated product, Compound 103, (yield: 175 mg, 50%) ischaracterized by TLC (R_(f)=0.35 in chloroform/methanol/ammonia(90:9:1)) and by MS ([M+H]+=602).

To a solution of 150 mg of Compound 103 (0.25 mmol) in 2 ml of dryacetonitrile are added 50 μl of diethanolamine (0.5 mmol, 2 eq.). Themixture is stirred at room temperature for 1 h. After removal of thesolvent the crude mixture is purified by flash chromatography. Thecollected fractions are concentrated under vacuum and yield a yellowishsolid (60 mg, 40%). The expected compound 104 is characterized by TLC(R_(f)=0.15 in chloroform/methanol/ammonia (90:9:1)) and MS([M+H]+=579).

To a mixture of 800 mg glutaric acid (6 mmol, 6 eq.) and 500 mg CDI (3mmol, 3 eq.) dissolved in 10 ml of dry acetonitrile, which is stirredfor 30 minutes at room temperature under argon, are added 266 mg AZT (1mmol) and a catalytic amount of DMAP. The cloudy reaction mixture isstirred overnight at 70° C. Chromatography yields a colorless stickysolid (120 mg, 35%). The expected compound 105 was characterized by TLC(R_(f)=0.25 in chloroform/methanol/ammonia (90:9:1)) and MS([M+H]+=381).

To a cloudy solution of 55 mg of Compound 105 (0.15 mmol, 3 eq.), and 29mg of Compound 104 (0.05 mmol, 1 eq.), 38 mg BOP (0.18 mmol, 3.3 eq.)and a catalytic amount of DMAP in 0.5 ml of dry THF are added 30 μl oftriethylamine (0.25 mmol, 4 eq.). The clear solution is then stirredovernight at room temperature. The mixture is purified bychromatography. The collected fractions yielded to a light yellowishsolid (5 mg, 10%). The expected double acylated Compound 101 ischaracterized by TLC (R_(f)=0.25 in chloroform/methanol/ammonia(90:9:1)) and by MS ([M+H]+=1306).

This protocol can be applied to other alcohols, some of which are listedin Table 3.

ABBREVIATIONS

DMAP=4-(N,N-dimethylamino)pyridine

BOP=(Benzotriazol-1-yloxy)-tris-(dimethylamino)-phosphonium-hexafluorophosphate

CDI=Carbonyldiimidazole

Z-=Benzyloxycarbonyl—

Example 29 Compound 106

286 mg of Celecoxib (7501 mmol), 300 mg of succinic anhydride (3 mmol, 4eq.), and 50 mg of DMAP are dissolved in 8 ml of dry acetonitrile. 420μl (300 μg, 3 mmol, 3 eq.) of triethylamine are added, and the reactionmixture is stirred overnight. 3 ml 1M aqueous sodium hydroxide and 5 mlof THF are added to remove excess succinic anhydride, the mixture isstirred for 2 h. 180 μl of acetic acid (3.1 mmol) are added and themixture is evaporated to dryness. The resulting oil is suspended inethyl acetate. Diluted aqueous ammonia is added, and the aqueous phaseis separated and evaporated until the gas evolution ceases. ConcentratedHCl is added to obtain a yellow precipitate. The residue is dissolved inethanol, evaporated to dryness and dried at 30° C./0.01 mbar for 2 h.The yield of the resulting Compound 107 is 350 mg (93%), and can be usedfor the following step without further purification.

240 mg of Compound 107 (5001 mmol) are stirred together with 110 mg ofCDI (650 μmol, 1.3 eq.) in 8 ml of dry dichloromethane for 2 h. 300 mg(400 μmol, 0.8 eq.) of Compound 43 are added and the mixture is stirredfor an other 2 h. The mixture is subjected to chromatography afterevaporation to yield 80 mg (16%) of the desired product, Compound 106(MS: [M+H]+=1213).

Example 30 Compound 108

To a stirred solution of 1.12 g erythromycin A oxime (1.5 mmol) in 50 mlTHF was added 1.5 ml 1 N potassium hydroxide solution (1.5 mmol) and0.44 g 4-bromomethyl-6,7-dimethoxycoumarin (1.5 mmol). The reactionmixture was stirred at room temperature for 6 h and then filtered andtreated with 44 μl of acetic acid. The solvent was removed under reducedpressure and the residue purified on silica gel, eluting withCHCl₃/MeOH/NH₄OH (6:1:0.1) to afford 0.4 g (28%) of Compound 108 acolorless foam. (MS: [M+H]+=968).

Compound 109

To a stirred suspension of 0.46 g (6,7-dimethoxy-2-oxo-2H-chromen-4-ylmethylsulfanyl)acetic acid (1.5 mmol) in 20 ml of dry CH₂Cl₂ are added250 mg N,N′-carbonyldiimidazole (1.55 mmol). The reaction mixture isstirred for 2 h, then a solution of 1.0 g Compound 43 (1.3 mmol) in 10ml of dry CH₂Cl₂ is added and stirring continued for 48 h. A saturatedaqueous solution of sodium bicarbonate (30 ml) is added. The organiclayer is dried over Na₂SO₄, filtered and then concentrated under reducedpressure to furnish the crude product. Chromatography affords theCompound 109 as a white foam (0.7 g, 52%). (MS: [M+H]+=1042).

Example 31 Compound 110

Imatinab may be selectively altered without compromising the interactionwith the kinase and thus its biological activity (Schindler et al.,Effects of a selective inhibitor of the Abl tyrosine kinase on thegrowth of Bcr-Abl positive cells. Science 289, 1938-1942, 2000).

2.2 g of 4-(4-Chlorocarbonyl-phenyl)-piperazine-1-carboxylic acid9H-fluoren-9-ylmethyl ester and 1.15 g4-Methyl-N3-(4-pyridin-3-yl-pyrimidin-2-yl)-benzene-1,3-diamine (U.S.Pat. No. 5,521,184) are reacted in 50 ml of dimethylformamide in thepresence of 600 mg dimethylaniline for 24 h. The mixture is poured into250 ml of ice-cold water. After filtration, the crude product is driedin vacuo and treated with a mixture of methanol and triethylamine(10:1). After evaporation of the solvent, the residue is subjected tochromatography to yield Compound 111.

40 mg of Compound 111 are dissolved in 2 ml dry ethanol at 60° C. andreacted with 31 mg of Compound 111 for 10 h. The mixture is cooled to−21° C. and filtered. The product, Compound 110, was obtained afterrecrystallisation. (MS: [M+2H]²⁺=628).

Biological Methods Example 32 Proliferation Assay

Assay to determine the in vitro rate of, for example, lymphocyteproliferation. Lymphocytes are purified out of ant coagulated (CPDA,citrate or heparin) mammalian blood using the Lymphoprep™ system(supplier). Purified cells are counted using a hemocytometer followingTrypan Blue staining, and a cell concentration of 1×10⁶ cells/mlestablished in RPMi 1640 medium with 10% FCS and antibiotics as required(all from Biochrome). Following the addition of a cell proliferationstimulant, for example phytohemagglutanin (Sigma) at, for example an endconcentration of 5 μg/ml, the cells are incubated with differentconcentrations of the to be investigated compound in 100 μl end volumein a 96-well microtiter plate in an incubator (37° C., 5% CO₂, 95%humidity) for 72 h. Cell proliferation is quantified following BrdUincorporation for 16 h by ELISA and subsequent calorimetric development(Cell Proliferation ELISA BrdU (colorimetric) kit from RocheDiagnostics). The IC₅₀ (μM) values are then calculated, and used tocompare compound efficacy.

To determine the influence of the T-L-C modification on in vitrocellular drug uptake and pharmacology, the above assay is additionallymodified and an additional “wash” step included. In addition to runningthe assay for 72 h with the compounds to be tested, the assay is alsorun for just 2 h, then compound is washed away in three serial washingsteps using 200 μl of medium at each step, and the cells subsequentlyincubated for a further 70 h. The determined IC₅₀ (IM) values following2 h and 72 h incubation are compared and a ratio calculated (2 h:72 h).The lower the number, the better the uptake and drug release from theT-L-C in the cells (see results in Table 4 for examples), andimprovement over mycophenolic acid.

TABLE 4 Proliferation assay results of T-L-C conjugates of mycophenolicacid IC₅₀ IC₅₀ Ratio Conjugate (μM) at 2 h (μM) at 72 h (2 h:72 h)Mycophenolic acid 2.5 0.54 4.63 Mycophenolate 1.5 0.33 4.5 mofetilCompound 67 1.74 1.36 1.3 Compound 79 3.16 1.2 2.63 Compound 74 3.2 2.21.45 Compound 80 1.41 0.4 3.53 Compound 81 1.78 1.12 1.6 Compound 692.66 1.4 1.9

Example 33 Cell-Based IMDPH Assay with Guanosine Rescue

Cytotoxicity Assay

HeLa cells (DSMZ, ACC 57) and Jurkat cells (DSMZ, ACC 282) inexponential growth phase are exposed for 3 days to test compounds. Thenumber of surviving cells is then determined by the Alamar Blue assay(Serotec Inc.). This assay incorporates a fluorometric growth indicatorbased on detection of metabolic activity. Specifically, the systemincorporates an oxidation-reduction indicator that fluoresces inresponse to chemical reduction of the growth medium resulting from cellgrowth. As cells grow in culture, innate metabolic activity results in achemical reduction of the immediate surrounding environment. Continuedgrowth maintains a reduced environment while inhibition of growthmaintains an oxidized environment. Reduction from growth causes theRedox indicator to change from an oxidized to a reduced form.Fluorescence is monitored at 560 nm (Exc.) and 590 nm Em.

General Procedure:

HeLa cells (1×10³) or JURKAT cells (1×10³) are plated in 100 μl MEMmedium (with Earle's salt; Biochrom KG) containing 10% FBS, 2 mML-glutamine, and non-essential animo acids in 96-well plates andincubated at 37° C. and 5% CO₂ atmosphere. After 24 hours, the testcompounds are added over a concentration range and the cells incubatedfor a further 48 hours. Alamar Blue reagent (20 μl) is added to eachwell, and the cultures incubated for a further 4 to 6 hours. Thefluorescence is then measured as described above and the LD₅₀ isdetermined based on a sigmoidal dose response regression. In order todetermine the toxicity of T-L-C conjugates of mycophenolic acid not dueto the inhibition of IMPDH, excess guanosine is added into the culturemedium to a final concentration of 50 μM. Any toxicity still detectedcan then be ascribed either to other biological effects of the of T-L-Cconjugate of mycophenolic acid, or is due to the very high intracellularconcentration of mycophenolic acid, following concentrative uptake intothe cell.

Cytotoxicity Assay with Fresh PBMNCs

The cytotoxicity of T-L-C conjugates of mycophenolic acid can bedemonstrated directly on freshly isolated mammalian PBMNCs. The cellsare prepared as described in Example 29, and the level of cytotoxicitydetermined by the Alamar Blue assay, as described above. As describedfor both HeLa and JURKAT cells, guanosine can also be used here toameliorate the effect of mycophenolic acid on the activity of IMPDH.

Results:

The toxicity of mycophenolic acid conjugates may be assessed mostconveniently in a cell based system, preferably with a rapidly growingcell line such as HeLa or JURKAT. In normal culture conditions,mycophenolic acid has an IC₅₀ of less than 2 uM, and its effect can becompletely removed in the presence of 50 μM guanosine. For many of theT-L-C conjugates of mycophenolic acid, alleviation with guanosine ispossible, but this is not always complete, which could for example bedue to either to other biological effects of the of T-L-C conjugate ofmycophenolic acid, or is due to the very high intracellularconcentration of mycophenolic acid, following concentrative uptake intothe cell.

Example 35 Efficacy Testing of Immunosuppressive Drugs Using a MouseSkin Transplant Model

Skin transplant rejection is a strong immune response and serves as avery sensitive test of the immunosuppressive potential of drugs in organtransplantation and graft rejection. The mouse trunk skin transplantmodel was established using published methods (Dillingham et al., 1954).Donor (Bl 10) trunk skin (approximately 8×8 mm) is removed and kept coldin saline before grafting on recipient Balb C mice. Male mice (n=10) aredosed orally once a day from the day of transplant surgery (day 0) untilthe day of rejection. For each study, appropriate vehicle-treatedcontrol groups are run concurrently. Graft rejection is quantified asthe number of days to reach R4 rejection (>75% of graft scabbed).

Results:

An example of results obtained with T-L-C conjugates of mycophenolicacid in the mouse skin transplant model are shown in FIG. 9. The meanrejection time for the vehicle, in this experiment saline, was 11.8days, while the rejection time of the T-L-C conjugate Compound 67 was13.5 days. Treatment with Compound 67 using dosage tapering (FIG. 10),resulted in a mean rejection time of 13.4 days.

Example 36 Testing of Antibiotic Activity of Drugs Assay Summary

The TC₅₀ or MIC procedure for antibiotic sensitivity testing involves anantibiotic dilution assay, which can be performed in microtitre plates.A series of twofold dilutions of each antibiotic are made in the wells,and then all wells are inoculated with a standard amount of the sametest organism. After incubation, growth in the presence of the variousantibiotics is observed by measuring turbidity. Antibiotic sensitivityis expressed as the concentration of the antibiotic that inhibits 50% ofthe growth (TC₅₀). Alternatively it could be expressed as the highestdilution of antibiotic that completely inhibits growth (MIC).

Bacteria: B. pumilus and E. coli (DH5α)Bacterial cultures are initiated from the plates for 2 to 3 weeks. Afterthis time period bacteria are streaked out on new plates from thebackups stored at −80° C. Due to the lack of resistance of the bacteria,new cultures are not to be initiated from an old plate or any liquidcultures derived from old plates.Growth medium (GM) (per liter): 10 g Bacto-tryptone, 5 g Bacto-yeastextract, 6 g HEPES (25 mM), 5.4 g NaCl, pH 7.3Compound stocks 10 or 100 mM in DMSO stored at −20° C.

Procedure

-   1. Grow B. pumilus from an LB agar plate in a flask (max. 10%    volume) up to about 50 ml in growth medium (GM)-   2. Dilute overnight suspension 1:10 in GM-   3. Determine OD₆₀₀ of diluted bacterial suspension-   4. Dilute bacterial suspension in GM to an OD₆₀₀ of 0.03-0.04. (6    ml/plate)-   5. Add 200 μl GM to the outer wells (Row A, Row H, Column 1, Column    12)-   6. Add 100 μl GM to each well starting from C2, row 3.-   7. Controls: Wells B2-B4 growth control. Wells B6-B8 blank. Row C    growth inhibition control.    -   7.1. To wells B2-B4 add: 96 μl GM, 4 μl DMSO, and 100 μl        bacterial suspension adjusted to an OD₆₀₀ of 0.03-0.04.    -   7.2. To wells B6-B8 add: 196 μl GM and 4 μl DMSO    -   7.3. Dilute a 10 mM Compound 43 (positive control) stock to 800        μM (120 μl/plate) in GM. Add 100 μl of 800 μM Compound 43        solution to well C2.-   8. Samples    -   8.1. Dilute the 10 or 100 mM stock solutions to 800 μM (250        μl/plate) in GM.    -   8.2. Add 100 μl of 800 μM sample in duplicates to wells D2/E2        resp. F2/G2.    -   8.3. 2-fold serial dilution of all samples and Azithromycin    -   8.3.1. Rows C-G, Columns 2: Mix and transfer 100 μl from each        row to Column 3, and continue until column 11. The remaining 100        μl out of column 11 are disposed.-   9. Add 100 μl of bacterial suspension (OD₆₀₀ 0.03-0.04) to each well    from C2-G11.-   10. Incubate plates on shaker, 750 rpm, 37° C., until the growth    controls have reached an OD_(600 of) 0.6-0.8 (approximately 6-8 h).

11. Determine OD₆₀₀ on plate reader.

TABLE 5 TC₅₀ values for representative compounds. Compound TC₅₀ in E.coli (μM) COMPOUND 43 2.3 COMPOUND 40 >50 COMPOUND 96 28 COMPOUND 53 >50COMPOUND 45 >50

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

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1. A method of identifying a compound useful for enhancing efficacy of atherapeutic agent, comprising incubating a compound in blood cells,separating immune cells from erythrocytic cells, and determining theratio of the concentration of the compound in the immune cells to theconcentration of the compound in the erythrocytic cells, wherein thecompound comprises a transportophore and a therapeutic agent, in whichthe transportophore is covalently bonded to the therapeutic agent via abond or a linker.
 2. The method of claim 1, the compound has thefollowing formula:TL-C)_(m), wherein T is a transportophore, L is a bond or a linkerhaving a molecular weight up to 240 dalton, C is a non-antibiotictherapeutic agent, and m is 1, 2, 3, 4, 5, 6, 7, or 8, in which thetransportophore has an immune selectivity ratio of at least 2, thetransportophore is covalently bonded to the non-antibiotic therapeuticagent via the bond or the linker, and the compound has an immuneselectivity ratio of at least
 2. 3. The method of claim 1, wherein theseparating is by density gradient centrifugation.
 4. The method of claim1, wherein the determining is by fluorescence microscopy.
 5. The methodof claim 1, wherein the determining is by liquid chromatography.
 6. Themethod of claim 1, wherein the therapeutic agent is an anti-inflammatoryagent.
 7. The method of claim 1, wherein the therapeutic agent is ananti-infectious agent.
 8. The method of claim 1, wherein the therapeuticagent is an anti-cancer agent.
 9. The method of claim 1, wherein thetherapeutic agent is an allergy-suppressive agent.
 10. The method ofclaim 1, wherein the therapeutic agent is an immune-suppressant agent.11. The method of claim 1, wherein the therapeutic agent is an agent fortreating a hematopoietic disorder.
 12. The method of claim 1, whereinthe therapeutic agent is an agent for treating a metabolic disease. 13.A method for delivering a therapeutic agent with a selectiveconcentration, comprising: identifying a compound by a method of claim1, and delivering the compound to a cell, wherein the compound comprisesa transportophore and a therapeutic agent, in which the transportophoreis covalently bonded to the therapeutic agent via a bond or a linker.14. The method of claim 13, wherein the cell is a cell of respiratorytissue.
 15. The method of claim 13, wherein the cell is a cell ofneoplastic tissue.
 16. The method of claim 13, wherein the cell is acell mediating allergic responses.
 17. A cell comprising a therapeuticagent identified by the method claim
 1. 18. A cell comprising atherapeutic agent identified by the method claim 2.